www scbt com protocols

Protocol for ELISA Kit from Biorbyt: Tools for Science FOR RESEARCH USE 6221 4503 0 www.scbt.com BACKGROUND Fibronectin is an extracellular. See our web site at www.scbt.com for detailed protocols and support products. RESEARCH USE. For research use only, not for use in diagnostic procedures. You can also access our most popular protocols straight from your phone with the Abcam app, which features protocols, scientific support and a suite of.
www scbt com protocols

Www scbt com protocols -

StatusPublic on Mar 30, 2018TitleMM231 cells tfxn LSD1 shRNA, biological rep1Sample typeRNA Source namebreast cancer cell line
OrganismHomo sapiensCharacteristicscell line: MDA-MB-231
cell type: breast cancer cell line
transfected with: LSD1 shRNA
Treatment protocolshRNA Lentiviral Particles were purchased from Santa Cruz Biotecnology and transduced according to manufacturer's protocol (http://www.scbt.com/protocols.html?protocol=shrna_lentiviral_particles_transduction)
Growth protocolnormal culturing conditions for MDA-MB-231
Extracted moleculetotal RNAExtraction protocolQIAgen RNeasy Mini (Animal Cells Spin protocol, w/DNase I digest performed on homogenized lysate prior to applying lysate to column for purification)
Labelbiotin
Label protocolcRNA synthesis and labeling was performed using the Ambion MessageAmp Premier Kit (Life Technologies)
 Hybridization protocolcRNA was hybridized to U133A 2.0 arrays (Affymetrix, Santa Clara, CA)
Scan protocolcRNA synthesis and labeling, hybridization, and scanning were performed by the University of Pittsburgh Cancer Biomarkers Facility
DescriptionMDA-MB-231 cells, LSD1 shRNA transfection, biological replicate 1
Data processingThe data is RMA normalized using 'affy' package in R-Bioconductor
 Submission dateJan 03, 2018Last update dateMar 30, 2018Contact nameYi HuangOrganization nameUniversity of Pittsburgh Cancer Institute
DepartmentPharmacology & Chemical Biology
LabMWRI, B430I/J
Street address204 Craft Ave
CityPittsburghState/provincePennsylvaniaZIP/Postal code15213CountryUSA Platform IDGPL571Series (1)
GSE72687RNA expression in MDA-MB-231 cells transfected with scramble, LSD1 or HDAC5 shRNA (HG-U133A_2)
Источник: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM2911381

Santa Cruz Biotechnology, Inc.

USDA Enforcement Actions

USDA Hearings Against SCBT

Inventories

The following figures were obtained from animal inventories at SCBT, as documented by USDA veterinary inspectors while conducting compliance inspections. AWI believes it likely that the dramatic change is related to two key events in August 2015: the USDA filing an unprecedented third complaint for violations of the Animal Welfare Act against SCBT and the USDA presenting a strong case demonstrating violations by the company before an administrative law judge. For further details see AWI's February 2016 press release.

DATERABBITSGOATS
April 22, 201430003400
November 6, 201430002660
July 7, 201524713202
September 23, 201517140
January 12, 201600

USDA Inspection Reports

Santa Cruz Biotechnology, Inc., operates as both a USDA registered research facility and a USDA licensed dealer. Inspections on each of the dates below include the reports for SCBT as both a dealer and a research facility (the certificate number 93-B-0192 in the upper right corner designates the dealer and 93-R-0380 designates the research facility).

May 5, 2010

July 13, 2010

February 8, 2011

April 5, 2011

October 4, 2011

March 6, 2012

April 19, 2012

May 2, 2012

May 24, 2012

June 26, 2012

August 23, 2012

September 26, 2012

October 31, 2012

December 18, 2012

February 20, 2013

May 14, 2013

September 10, 2013

April 22, 2014

November 6, 2014

July 7, 2015

September 23, 2015

January 12, 2016

AWI Requests Action from Federal Agencies

SCBT: Supplying Antibodies Is Big Business

For years, SCBT has been one of the world's largest suppliers of antibodies produced in animals. A survey published in 2007 by Biocompare ranked it as the “top company” for all types of antibodies purchased. Another survey published in 2009 by Bioinformatics also ranked it as the largest. In 2012, a survey published in The Scientist ranked SCBT a close second among 15 of the biggest suppliers globally, while another 2012 survey by BioAstrum ranked the company second in the number of citations published in scientific journals in 2011.

USDA Hearings Against SCBT: August 2015

The following are AWI's notes from the administrative hearing brought by the USDA against SCBT for apparent violations of the Animal Welfare Act.  The hearing was held before Judge Janice Bullard from August 18-21, 2015.

Tuesday, August 18, 2015
The hearing for the USDA's case against Santa Cruz Biotechnology, Inc. (SCBT) before Administrative Law Judge Janice Bullard began today. Colleen Carroll is the attorney from the USDA's Office of General Counsel and Mark Lynch; Dr. Jeannie Perron, and Cortlin Lannin are attorneys with Covington & Burling, the extremely powerful law firm representing SCBT.

The hearing began with a variety of housekeeping matters, but we learned that the USDA intends to call a “mystery witness,” who is a former employee of SCBT. The person has not wanted to be identified because of a concern that there would be pretrial retaliation and the person is worried for her safety. This individual has been subpoenaed to testify, and it is expected she will do so later this week.

The first person to testify was Dr. Carol Clarke, Research Program Manager with the USDA's Animal Care program. She testified about an inspection of SCBT she participated in on March 6, 2012, where she observed a number of goats in poor condition. She described one goat with large areas of hair loss and scabby lesions and another goat who was lame and extremely thin. Dr. Clarke described the process of extracting blood for antibody production and the need to be vigilant with the animals' health and condition if they are being bled on a regular basis. She described SCBT's failure to monitor the condition of its goats to ensure their welfare.

Dr. Laurie Gage testified next. Although she is USDA's big cat and marine mammal specialist, the title doesn't accurately represent all the work she has done for the department. She participated in an inspection of SCBT on July 13, 2010. She stated that she saw a number of animals “in distress or pain or suffering.” One was a goat with a “nasty, fairly fresh wound on its hind leg” that she was told was probably from a coyote attack. There were two debilitated goats: One was put out in the hot sun by an untrained worker; this goat was unable to move on his/her own to the shade. Another was a goat lying in the food bunker, also unable to move. Though a staff member put food nearby, it was out of reach. However, when the inspector presented food within reach, the goat ate it.

The final witness for the day was Dr. Marcy Rosendale, a veterinary medical officer with the USDA's Animal Care program since 2003. She has conducted inspection after inspection of SCBT from 2003 right up to the most recent inspection conducted last month. She spent the afternoon describing all of the atrocities she had seen. When asked about the facility's ability to comply with the law, Dr. Rosendale replied, “I don't see a will. Others do it, but I'm not seeing a willingness to come into compliance.” She described seeing “the same problems over and over and over again” at SCBT.

Dr. Rosendale described how various employees of SCBT had denied the existence of another location with a barn and additional animals farther down the road from the main area. Even when the employees were asked directly the existence of this other location, they said no, there wasn't one. Then, later, Dr. Rosendale described a call she received from a past employee of SCBT who apologized for denying there was an additional location with more animals. The former employee said there were 1,300 goats at this other location—that the USDA had not been informed of and had not inspected.

Dr. Rosendale will be cross-examined by SCBT's attorneys tomorrow.

Wednesday, August 19, 2015
The hearing resumed with SCBT's attorney, Mark Lynch, cross-examining Dr. Rosendale, which was followed by redirect from Colleen Carroll. When questioned about whether she inspects other antibody production facilities, Dr. Rosendale said that she inspects two others that also have goats and rabbits; in responding to a crucial follow-up question from Ms. Carroll, Dr. Rosendale said that these other facilities do not have the same deficiencies she has found at SCBT.

Mr. Lynch moved on to ask Dr. Rosendale about her conversation with Dr. Robin Parker, who had been SCBT's veterinarian. After she left SCBT, Dr. Parker contacted Dr. Rosendale to inform her of an additional facility that Dr. Parker said had been concealed from the USDA. This site was referred to as Barn H7. Dr. Rosendale recounted how upset Dr. Parker was by what was going on at SCBT, and she recalled that Dr. Parker was crying during one inspection when one of the goats died in front of them. Dr. Parker told Dr. Rosendale that she felt so overworked, working 70-80 hours a week with no relief, that she thought she would have a nervous breakdown, and her own doctor put her on medical leave. She was fired while on leave before she could resign.

In response to a question from Judge Bullard, Dr. Rosendale emphasized that the USDA did NOT know about the other (H7) facility prior to hearing about it from Dr. Parker. Most importantly, Dr. Rosendale was able to establish that she asked on many occasions (as was customary) and in different ways if there were other facilities that should be inspected, and she was always told “No.” She added that Dr. Parker “remembered me asking [if there were other facilities that should be inspected] and felt guilty” for not telling her about the undisclosed H7/Lake Ranch barn, which was located several miles from the rest of the compound.

Dr. Rosendale also testified to the inadequacy of the staffing levels for the number of animals at SCBT; that as the number of animals increased, so did the number of problems; and that when she would return to check on noncompliant items from a previous inspection, there would be new noncompliant items.

The last witness on Wednesday was Dr. Pamela Smith, a veterinary medical officer (VMO) for Animal Care in the San Francisco Bay area. She testified that she accompanied Dr. Rosendale on 13 inspections of SCBT and found a recurring theme: animals in poor condition for whom it seemed nothing was being done. There were no notes in the records that a problem existed, much less that anything was being done. She recalled the inspection that involved a goat with the broken leg; she characterized as “pretty shocking” and “quite memorable” the fact that this animal hadn't received any veterinary care. The next inspection was also “pretty shocking”: This was when the “goat died in front of us.”

Dr. Smith also testified that SCBT employees were repeatedly asked whether there were other facilities that should be inspected, and that those employees repeatedly said there were not. When they finally did find out about barn H7/Lake Ranch from Dr. Parker and asked SCBT staff yet again about another site, they were told, “well, there might be one” but it was difficult to get to; additionally, the SCBT vet, Dr. Naomi Weinberg—even though she said she had never been there—found it with no problem. Dr. Smith said Dr. Weinberg was “sheepish and uncomfortable” because “she knew we knew.”

Dr. Smith said that at the previously undisclosed barn they found elderly goats with a lot of health problems; there were “a lot of citations” as a result. Ms. Carroll asked specifically about the cause of nasal discharge in several goats, and Dr. Smith said it was likely a viral respiratory infection with a secondary bacterial infection.

Ms. Carroll then asked Dr. Smith's opinion of the effectiveness of the Institutional Animal Care and Use Committee (the oversight body at research facilities, mandated by law). Dr. Smith had several concerns about SCBT's IACUC. She said the IACUC should have been discussing the inspection reports and should have been having “substantive discussions about the repeated problems,” but there was no evidence of that. She had the impression that the Committee was “not that engaged.” Moreover, the community member1 of the IACUC was a neighbor who, after repeated attempts by USDA to contact him, resigned from the Committee.

At that point, Dr. Smith said that the IACUC was not properly functioning and should not have approved changes in any of the protocols, but it did so anyway. Dr. Smith also discovered goats with lesions and swelling in their shoulders from injections, when the protocol called for them to be injected in the ribcage. The SCBT vet, Dr. Warden, contended that since the scapula is on top of the ribcage, that's still the ribcage. To Dr. Smith, not only was this procedure contrary to the protocol, but the swelling was indicative of the employees not doing it properly, thereby making it a health concern.

Thursday, August 20, 2015
The first to testify was the “mystery witness.” She is Dr. Robin Parker, who worked as a veterinarian for SCBT beginning in early 2011. In addition to her responsibilities for the approximately 11,000 goats and 6,000 rabbits at SCBT during her tenure, a primary responsibility was to care for about 100 horses at the ranch and a herd of about 200 Gelbvieh cattle owned by John and Brenda Stephenson (who also own SCBT). Her responsibilities expanded still further with the launching of a new venture by the Stephensons; Santa Cruz Animal Health, distributing prescription drugs to veterinarians. This office was 35 miles away.

Within the first month after starting her job at SCBT, Dr. Parker said she was told about a location at the facility that was undisclosed to the USDA, and she was instructed that the situation needed to remain that way. While the employees at SCBT were aware of this location and that the animals there were being bled for antibody production, she said she was told that SCBT didn't want the USDA to know about it because SCBT was trying to minimize the potential for further citations by the inspectors. Dr. Parker said the company CEO, John Stephenson, was the one who made the decision not to have the location revealed. It was felt by Dr. Stephenson that the USDA tended to nitpick on its inspections of SCBT. Later, under cross-examination by SCBT attorney Mark Lynch, Dr. Parker was asked if she felt that USDA was nitpicky and she replied, “No sir, I don't. I would be hard-pressed to say that's true.”

Dr. Parker described some of her specific concerns with SCBT. Bleeds were conducted on animals without determining their eligibility by weighing them or checking their hematocrit levels1 to ensure that the appropriate amount of blood was taken and that the animals were remaining stable. While the lab claimed that it had a number of veterinary technicians on staff, none were actually registered as such, which Dr. Parker said is mandated by the State of California. Dr. Parker stated that lack of training was an issue with SCBT staff. The member on the IACUC serving as the “unaffiliated member” mandated by the AWA to represent “general community interests in the proper care and treatment of animals” was the real estate agent who had been instrumental in the purchase of the company property.

Dr. Parker said that there was a lack of shade for the animals and the temperatures in summer would exceed 100 degrees. The feeders used for the goats were actually intended for cattle and therefore they did not work well for the goats even when SCBT tried removing the legs on the feeders. Goats climbed in them and urinated and defecated. The goats would also get stuck in them and when the feeders were originally set up, hay particles would fall into the animals' eyes, creating irritation and conjunctivitis. Eventually, some proper feeders were acquired, but only for use by those goats in the hospital facility, not at any other locations.

Under questioning by USDA attorney Colleen Carroll, Dr. Parker acknowledged that she was present on at least one occasion when a goat was agonal (exhibiting gasping, labored breathing) and euthanasia was called for. In fact, the goat died before she was able to euthanize it. When asked if there were animals that needed veterinary care and were agonal, she replied, “Yes.”

After 14 months, Dr. Parker took medical leave from her job, and she was terminated before she returned to work.

USDA rested its case.

The first called to testify for SCBT was Dr. Robert Peterson of Case Western Reserve University in Cleveland. He defended the manner in which SCBT used Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant2 based on his reading of documents about the facility, including its Standard Operating Procedures, protocols, photographs, and USDA inspection reports. He stated that CFA is accepted by the research community, and that while it is not ideal, “it is the gold standard.”

Under cross-examination by Ms. Carroll, Dr. Peterson stated that he has never been to SCBT. It appeared that he was unaware that they make their own CFA, as most places don't have the capacity to do so. When asked if the condition of the animal enclosures could be a factor with bacterial lesions, he replied, “Yes.” When asked whether, if bacteria are in the CFA, it could be transmissible to rabbits, he said, “Yes.” When asked if this could cause granulomas, he again replied, “Yes.”

Dr. Peterson explained the attention needed for animals who are being bled, the importance of looking at the weights of animals and their condition, including the condition of their coats, and that as a prey species, goats and rabbits may not show pain—rather, they hide it. At Case Western these animals would be assessed daily.

Finally, when asked about the use of specific-pathogen free (SPF) rabbits in antibody production, Dr. Peterson appeared unsure. He said that Case Western purchases and uses SPF rabbits for antibody production. Based on this line of questioning, it seems that the rabbits SCBT uses for antibody production are not SPF.3

The final witness for the day was Dr. Don Warden, SCBT's attending veterinarian. He first began doing work for the facility in 2003, and then he became their attending vet in either 2005 or 2007 (he couldn't recall which). He talked about the great working relationship he had with the staff and that he is viewed as a friend and that they have no reservations in coming to him with any issues.

SCBT attorney Cortlin Lannin began a process of going methodically through the animals identified by the USDA to be of concern and Dr. Warden explained away most of them. For example, he felt it was not possible that a staff member would have placed a goat that wasn't ambulatory out in the sun; the animal must have gotten out there on its own. Or those goats that choose to go in the feed bunker do so because it's “a nice cozy place to lie down and eat their food.” Where the USDA expressed concern that animals were lame or thin or experiencing respiratory conditions, Dr. Warden would explain that he was taking time to try to help the animals recover and just keep an eye on them. Regarding a recumbent animal he was asked about, Dr. Warden first explained that the term just means the animal is lying down. However, he acknowledged that if it was prodded and didn't get up, then you have a problem. In this case the goat “went to a corner because it didn't feel well. It cuddled up in there.” However, he did add that in hindsight he would have handled this particular goat differently.

At 6:00 p.m. Judge Bullard suggested that questioning of Dr. Warden be resumed in the morning. The judge noted that she would have some questions specifically about the veterinary records and wondered if Dr. Warden would be the one able to answer them or if someone else was going to testify about the records. She said there appeared to be no name associated with a number of records, many of them were not in chronological order, and there were other issues.

Friday, August 21, 2015
Prior to the start of today's proceedings, attorneys representing both sides exited the hearing room to converse privately. After approximately 45 minutes to an hour, the parties reentered the hearing room, at which point, Judge Bullard announced that the hearing would be suspended and that September 30, 2015, is the last day for parties to file a status report about their position regarding the reinstatement of an ongoing hearing. Please note that a settlement was not reached and the hearing is scheduled to resume on April 5, 2016.

USDA Hearings Against SCBT: August 2016 (CANCELED)

The hearing which began on August 18, 2015, was suspended on its fourth day so settlement discussions could be pursued. At the end of September 2015, the USDA reported that the likelihood of a settlement was “remote” and requested a resumption of the hearing. The hearing, on the USDA's first and second complaints against SCBT, which was scheduled to resume on April 5, 2016, before Administrative Judge Bullard, has been postponed until August 15, 2016. Immediately following this hearing, another hearing, on the USDA's third complaint against SCBT, is set to begin.

This postponement, requested by SCBT and opposed by the USDA, is the fourth delay of proceedings against the company. In her March 18, 2016, order granting SCBT’s request, Judge Bullard wrote that she “agreed that the hearing should not be postponed indefinitely,” indicating the facility may have attempted to avoid setting a new hearing date altogether.

A chart, which shows the numerous delays of the USDA hearings against SCBT, can be seen here. Two of the four delays were described as attempts to reach settlement.

On May 19, 2016, a settlement agreement was reached between the USDA and SCBT, and therefore, the hearing was canceled. More information can be found here.

Related Content

Research Facility Gets Off Easy Despite Horrendous Animal Welfare Act Violations
AWI Quarterly Article, Spring 2017

Complaint Filed Against Research Facility Involved in Dozens of Monkey Deaths
AWI Quarterly Article, Winter 2016

Scientists Must Step Up After USDA Comes Down on SCBT
AWI Quarterly Article, Fall 2016

Santa Cruz Biotech Stalls as Reputation Falls
AWI Quarterly Article, Summer 2016

Researchers Recoil from Santa Cruz Biotech as Company Jettisons Its Goats and Rabbits
AWI Quarterly Article, Spring 2016

Santa Cruz Biotechnology Agrees to $3.5 Fine, License Revocation
AWI Press Release, May 2016

US government issues historic $3.5-million fine over animal welfare
Nature Article, May 2016

Biotech firm to pay $3.5M fine to settle animal-abuse case
Associated Press Article, May 2016

Disappearance of Goats, Rabbits at Santa Cruz Biotechnology Raises Questions About Facility's Future
AWI Press Release, February 2016

Thousands of goats and rabbits vanish from major biotech lab
Nature Article, February 2016

Scientists Are Boycotting This Company For Alleged Goat Abuse, Bad Tweets
BuzzFeed Article, February 2016

Источник: https://awionline.org/content/information-santa-cruz-biotechnology-inc

Antibody dilutions and titer

Which dilution to use

The rate of binding between antibody and antigen affinity constant can be affected by temperature, pH and buffer constituents. Varying the relative concentrations of an antibody and an antigen solution can also control the extent of antibody-antigen complex formation. As it is not usually possible to change the concentration of the antigen, the optimal working concentration of each individual antibody must be determined with dilutions for each application and set of experimental conditions.

Many of our antibodies have recommended dilutions for various applications included on the datasheet. However, they may require some optimization.

Search primary antibodies 

Optimizing the antibody dilution: titration experiments

The optimal antibody concentration, which gives the best staining with minimum background, must be determined experimentally for each assay and is usually determined by using a series of dilutions in a titration experiment. For example, if a product datasheet suggests using a 1:200 dilution, it is recommended to make dilutions of 1:50, 1:100, 1:200, 1:400 and 1:500.

A titration experiment is done by first selecting a fixed incubation time and then a series of experimental dilutions of the antibody. Each dilution should be tested on the same type of sample in order to keep the same experimental conditions.

Many antibodies will have similar batch-to-batch consistency, therefore in most cases only one titration experiment is required. However, especially for polyclonal antibodies, when there is a change in the results of the staining between batches of the same antibody, we recommend performing another titration experiment.

Suggested dilutions for antibodies with no recommended dilution on the datasheet

Unpurified antibody preparations differ significantly in antibody concentration. If the specific antibody concentration of a given unpurified antibody preparation is unknown, we recommend to use a concentration/purification kit and refer to the table below as a guideline.

This table provides various dilutions to use in each application from different sources of antibody:


Tissue culture supernatantAscitesWhole antiserumPurified antibody
WB/dot blot1/1001/10001/5001 µg/mL
IHC/ICCNeat –1/101/1001/50–1/1005 µg/mL
EIA/ELISA1/10001/100001/5000.1 µg/mL
FACS/Flow cytometry1/1001/10001/5001 µg/mL
IP-1/1001/50–1/1001–10 µg/mL
Approximate IgG concentration estimate1–3 mg/mL5–10 mg/mL1–10 mg/mL-


​Return to the antibody guide.

You can also access our most popular protocols straight from your phone with the Abcam app, which features protocols, scientific support and a suite of useful tools that are handy for any bench scientist. Learn more.

Источник: https://www.abcam.com/protocols/antibody-dilutions-and-titer

The Nutcracker with Live Orchestra

Saturday & Sunday, Dec. 18-19, 2021 - 1 p.m. & 5 p.m.

Santa Cruz Civic Auditorium, 307 Church St., Santa Cruz, CA

From Santa Cruz Ballet Theatre:

"All performances with live orchestra!

"Santa Cruz Ballet Theatre presents the original Santa Cruz Nutcracker production, since 2002 at the Civic Auditorium with the Santa Cruz Ballet Theatre Orchestra conducted by Music Director Pamela Martin.

"We are excited to be back on the stage! No other production in the area features 52 professional musicians, bringing the beauty of Tchaikovsky's score to glorious life. 

"Revisit our favorite holiday tradition! Special guest stars and SCBT alumni Melody Mennite and Lucien Postlewaite will grace the stage as the Sugar Plum Fairy and her Cavelier.

"The SCBT Nutcracker features stunning choreography, costumes, scenery, lighting, right in the Civic Auditorium. Health and safety protocol will be in place for a safe viewing environment for everyone.

"All dancers, musicians, crew and audience must adhere to the Civic's Covid policies. 

"In accordance with California State (CDPH) requirements, all persons accessing the venue will be required to show proof of a COVID-19 vaccination (with matching photo ID) or negative PCR test taken within 72 hours (with matching photo ID). Mask wearing will also be mandatory until further notice. You will not be permitted to enter without these items.

"View the complete and current protocol HERE."

INFO & TICKET LINKS HERE

--Shutterstock image

Источник: https://patch.com/california/santacruz/calendar/event/20211218/1564809/the-nutcracker-2021-santa-cruz-civic-auditorium

Santa Cruz Biotechnology Protocols

Protocols
The Power to Question

Santa Cruz Biotechnology, Inc. provides an extensive range of high quality support products to complement our
broad range of primary antibodies. These include internally-produced secondary antibodies for Western blotting and
immunohistochemical applications, Luminol reagent, agarose-conjugated Protein A, Protein G PLUS and Protein L
for immunoprecipitation studies, nuclear extracts, whole cell lysates, tissue extracts, ready made blots for Western
blotting, Western blotting membranes, TransCruz™ gel shift oligonucleotides, blocking reagents and general lab sup-
plies. Cruz Marker™ Molecular Weight Standards and Prestained Molecular Weight Standards are provided for use
in Western blotting procedures. Our ImmunoCruz™ Staining System is a pre-diluted, ready-to-use system for immuno-
histochemical staining of paraffin-embedded tissue sections. We have also introduced a series of Apoptosis Detection
Kits for the assessment of cells undergoing apoptosis. The reagents and recommended procedures for use of our
products are provided below and include (1) Western (immuno-) blotting, (2) immunoprecipitation, (3) immunopre-
cipitation/Western blots, (4) ExactaCruz™ immunoprecipitation/Western blots, (5) immune complex protein kinase
assays, (6) immunoperoxidase cell staining, (7) immunofluorescence cell staining, (8) flow cytometry (FCM), (9) ELISA
assays, (10) TransCruz™ gel supershift assays, (11) methods for the use of peptides to neutralize antibody activity,
(12) ChIP assays, (13) siRNA transfection, (14) semi-quantitative RT-PCR and (15) preparation of solutions.

1. WESTERN (IMMUNO-)BLOTTING 䡲 Centrifuge cell lysate at 10,000xg for 10 minutes at


4° C. The supernatant fluid is the total cell lysate.
A. Sample Preparation Transfer the supernatant to a new microcentrifuge tube.
NOTE: For a listing of available cell culture products in- This is your whole cell lysate. For increased protein re-
cluding classical and specialty media, sera and media covery, resuspend the pellet in a small volume of RIPA,
additives, induction agents, antibiotics and attachment centrifuge and combine supernantants.
agents, please see our catalog or visit our website at NOTE: For phosphorylation studies, lysates can be
www.scbt.com. enriched for phosphoproteins using Santa Cruz Bio-
MONOLAYER CELLS technology Inc.’s PhosphoCruz™ Protein Purification
System (sc-24964).
䡲 Grow cells to subconfluency in a 100 mm x 20 mm
petri dish, remove culture medium and rinse cell mono- SUSPENSION CELLS
layer with room temperature 1x PBS (10X liquid PBS: 䡲 Collect approximately 2.0 x 10 cells by low-speed
7

sc-24946). The following steps should be performed centrifugation (e.g. 200xg) at room temperature for
on ice or at 4° C using fresh, ice cold buffers. 5 minutes. Carefully remove culture medium.
䡲 Add 0.6 ml of RIPA buffer (sc-24948) to the monolayer 䡲 Wash the pellet with PBS at room temperature and again
cells in the plate. Gently rock plate for 15 minutes at collect by low-speed centrifugation. Carefully remove
4° C. Remove adherent cells with a cell scraper. Trans- supernatant.
fer the resulting lysate to a microcentrifuge tube. 䡲 Add 1.0 ml of ice cold RIPA buffer (sc-24948) with freshly
䡲 Wash the plate once with 0.3 ml of RIPA buffer and added protease inhibitors and/or phosphatase inhibitors.
combine with first lysate. Gently resuspend cells in RIPA buffer with a pipet and
Optional: Add 10 µl of 10 mg/ml PMSF (sc-3597) stock incubate on ice for 30 minutes.
and/or pass through a 21-gauge needle to shear the 䡲 Further disrupt and homogenize cells by hydrodynamic
DNA.) Incubate 30–60 minutes on ice.

Santa Cruz Biotechnology, Inc. 1.800.457.3801 831.457.3800 fax 831.457.3801 Europe +00800 4573 8000 49 6221 4503 0 www.scbt.com
shearing (21-gauge needle), dounce homogenization 䡲 Load up to 10 µl of lysate per 1.0 mm of well width for
or sonication, taking care not to raise the temperature gels of 0.75 mm thickness.
of the lysate. (Optional: Add 10 µl of 10 mg/ml PMSF 䡲 We recommend the use of Cruz Marker™ molecular
stock. Incubate 30 minutes on ice. weight standards (sc-2035). Load 2 µl/well for 0.75 mm
䡲 Transfer to microcentrifuge tube(s) and centrifuge at gels and 5 µl/well for 1.5 mm gels. When used with
10,000xg for 10 minutes at 4° C. The supernatant fluid Cruz Marker™-compatible secondary antibodies, inter-
is the total cell lysate. Transfer the supernatant to a nal standard bands will appear when the probed blot
new microcentrifuge tube. This is your whole cell is exposed to detection reagent. Alternatively, use
lysate. For increased protein recovery, resuspend the Prestained Molecular Weight Standards (sc-2361).
pellet in a small volume of RIPA, centrifuge and com- 䡲 Electrophorese according to standard protocols.
bine supernantants.
䡲 Transfer proteins from the gel to a nitrocellulose or
NOTE: For phosphorylation studies, lysates can be PVDF membrane using an electroblotting apparatus
enriched for phosphoproteins using Santa Cruz Bio- according to the manufacturer’s protocols.
technology Inc.’s PhosphoCruz™ Protein Purification
NOTE: Ready-made blots of human or mouse whole
System (sc-24964).
cell extracts or nuclear extracts or mouse tissues are
TISSUE SAMPLES available as Cruz Blot Systems™.
䡲 Weigh tissue and dice into very small pieces using a
C. Immunoblotting
clean razor blade. Frozen tissue should be sliced very
thinly and thawed in RIPA buffer (sc-24948) containing 䡲 Block non-specific binding by incubating membrane
protease inhibitors and/or phosphatase inhibitors. Use in Blotto (either Blotto A or Blotto B; IgG-free BSA,
3 ml of ice cold RIPA buffer per gram of tissue. sc-2323, is recommended when using anti-bovine
secondary antibodies) for 30–60 minutes at room
䡲 Further disrupt and homogenize tissue with a dounce
temperature. Alternatively, the membrane may be
homogenizer or a sonicator, maintaining temperature at
blocked at 4° C overnight in a covered container,
4° C throughout all procedures. (Optional: Add 30 µl of
using Blotto without Tween-20.
10 mg/ml PMSF (sc-3597) stock per gram of tissue.)
Incubate on ice for 30 minutes. 䡲 If using a phospho-specific antibody, add 0.01% (v/v) of
each Phosphatase Inhibitor Cocktails A and B (sc-45044
䡲 Transfer to microcentrifuge tubes, centrifuge at
and sc-45045) to the blocking solution and the antibody
10,000xg for 10 minutes at 4° C. Remove supernatant
diluent to inhibit phosphatases.
and centrifuge it again. The supernatant fluid is the
total cell lysate. A longer centrifugation may be neces- 䡲 Incubate the blocked membrane in primary antibody
sary to obtain a clear lysate. diluted in Blotto for 1 hour at room temperature. (For
phospho-specific antibodies: Use Blotto B with 0.01%
NOTE: For phosphorylation studies, lysates can be
(v/v) of each Phosphatase Inhibitor Cocktails A and B
enriched for phosphoproteins using Santa Cruz Bio-
(sc-45044 and sc-45045.) Optimal antibody concentra-
technology Inc.’s PhosphoCruz™ Protein Purification
tion should be determined by titration. We recommend
System (sc-24964).
a starting dilution of 0.5–2.0 µg/ml. Wash membrane
B. Electrophoresis three times for 5 minutes each with TBST.
䡲 Mix sample (40-60 µg whole cell lysate, 10-20 µg 䡲 Incubate the membrane for 45 minutes at room temp-
nuclear extract, 10-20 µl transfected lysate or 10-20 erature with horseradish peroxidase (HRP) conjugated
ng purified protein per lane) with an equal volume of secondary antibody or alkaline phosphatase (AP) con-
2x electrophoresis sample buffer (sc-24945) and boil jugated secondary antibody, diluted to 1:500–1:2000
for 2–3 minutes. Unused samples may be stored at in Blotto. If high backgrounds are observed, secondary
-20° C. antibody should be diluted further (up to 1:20,000). If

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Cruz Marker™ molecular weight standards (sc-2035) 䡲 Pellet cellular debris by centrifugation at 10,000xg for
are used in the gel, the Cruz Marker™ compatible sec- 10 minutes at 4° C. Transfer supernatant to a fresh
ondary antibodies must be used in order to visualize microcentrifuge tube on ice.
standards with ECL. 䡲 Preclear lysate by adding 1.0 µg of the appropriate
䡲 Wash membrane three times for 5 minutes each with control IgG (corresponding to the host species of the
TBST and once for 5 minutes with TBS. primary antibody), together with 20 µl of appropriate
䡲 Incubate membrane in Chemiluminescence Luminol suspended (25% v/v) agarose conjugate (Protein A-
Reagent (sc-2048) according to Luminol data sheetor Agarose, Protein G-Agarose, Protein A/G-Agaroseor
visualize proteins using standard protocols. If luminol Protein L-Agarose). Incubate at 4° C for 30 minutes.
is used for visualization, an HRP-conjugated secondary 䡲 Centrifuge at 3,000 rpm (e.g. Eppendorf 5415D; approx-
antibody must be used. imately 1,000xg) for 30 seconds at 4° C.
2. IMMUNOPRECIPITATION 䡲 Transfer the supernatantor approximately 100–1000 µg
total cellular protein, to a microcentrifuge tube. Add
NOTE: This procedure may be used for cells labeled with
1–10 µl (i.e. 0.2–2 µg) primary antibody (optimal anti-
radioactive compounds such as amino acids or orthophos-
phate. (Radioisotope use and disposal should conform to body concentration should be determined by titration)
institutional and governmental regulations.) Cell labeling and incubate for 1–2 hours at 4° C. Alternatively, if
should be carried out in medium lacking the relevant antibody agarose conjugate is employed, add 20 µl
nonradioactive compound. Starving cells appropriately (i.e. 5 µl packed beads) and incubate at 4° C for 1 hour
prior to labeling is recommended. Incubate cultured cells to over-night with mixing; skip the next step.
(80-90% confluent monolayer in 100 mm cell culture plate 䡲 Add 20 µl of the appropriate agarose conjugate suspen-
or approximately 2–5 x 107 suspension cells in flask). sion (Protein A-Agarose, Protein G-Agarose, Protein
Example: Following starvation, remove culture medium A/G-Agarose or Protein L-Agarose). Cap tubes and in-
and replace with methionine-free medium containing 5% cubate at 4° C on a rocker platform or rotating device
dialyzed fetal calf serum and 100 µCi/ml 35S-methionine. for 1 hour to overnight.
Incubate 1 hour at 37° C. For some proteins a longer 䡲 Collect immunoprecipitates by centrifugation at
labeling period (up to 18 hours) is preferable. Wash 3,000 rpm (approximately 1,000xg) for 30 seconds at
cells with PBS as necessary to remove unincorporated 4° C. Carefully aspirate and discard supernatant.
35S-methionine. 䡲 Gently wash pellet 2–4 times with 1.0 ml RIPA buffer
NOTE: For a listing of available cell culture products in- (more stringent) or PBS (less stringent), each time re-
cluding classical and specialty media, sera and media peating centrifugation step above.
additives, induction agents, antibiotics and attachment 䡲 After final wash, carefully aspirate and discard super-
agents, please see our catalog or visit our website at natant and resuspend pellet in 40 µl of 2x electrophor-
www.scbt.com. esis sample buffer (sc-24945).
䡲 Add 1–3 ml ice cold RIPA buffer (sc-24948) to sub- 䡲 Boil samples for 2-3 minutes and subject to electro-
confluent cell monolayer and incubate at 4° C for 10 phoresis and autoradiography. Unused samples may
minutes. For suspension cells, add the RIPA buffer to be stored at -20° C.
washed cell pellet in a microcentrifuge tube.
Optional: After boiling, samples may be centrifuged to
䡲 Disrupt cells by repeated passage through a 21-gauge pellet the agarose beads followed by SDS-PAGE analy-
needle or sonication. Transfer to a microcentrifuge sis of the supernatant.
tube.
3. IMMUNOPRECIPITATION/WESTERN BLOTS
䡲 Optional: Wash cell culture plate with additional 1.0 ml
NOTE: For a listing of available cell culture products in-
ice cold RIPA buffer and combine with original extract.
cluding classical and specialty media, sera and media

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additives, induction agents, antibiotics and attachment 䡲 After final wash, aspirate and discard supernatant and
agents, please see our catalog or visit our website at resuspend pellet in 40 µl of 2x electrophoresis sample
www.scbt.com. buffer (sc-24945).
䡲 Prepare a total cell lysate as described under Western 䡲 Boil samples for 2–3 minutes. Load up to 5–10 µl of
blot procedure. sample per 1.0 mm well width for gels of 0.75 mm
thickness.
NOTE: For phosphorylation studies, lysates can be
enriched for phosphoproteins using Santa Cruz Bio- 䡲 Continue with electrophoresis and immunoblotting as
technology Inc.’s PhosphoCruz™ Protein Purification described under Western blotting procedure.
System (sc-24964). NOTE: Depending on the secondary antibody that is
䡲 Preclear whole cell lysate (optional step) as follows. used, 55 kDa and 27 kDa heavy and light IgG chains,
To approximately 1 ml of whole cell lysate or tissue respectively, of the primary antibody may be detected.
extract, add 0.25 µg of the appropriate control IgG These bands will be less pronounced if a primary anti-
(corresponding to the host species of the primary anti- body agarose conjugate is used in the above procedure
body), together with 20 µl of appropriate suspended or if ExactaCruz™ Reagents are used.
(25% v/v) agarose conjugate (Protein A-Agarose,
4. EXACTACRUZ™ IMMUNOPRECIPITATION/
Protein G-Agarose, Protein A/G-Agaroseor Protein L- WESTERN BLOTS
Agarose). Incubate at 4° C for 30 minutes.
䡲 Prepare a total cell lysate as described under Western
䡲 Pellet beads by centrifugation at 3,000 rpm (approxi- (Immuno-) blotting procedure.
mately 1,000xg) for 30 seconds at 4° C. Transfer
supernatant (cell lysate) to a new microcentrifuge
䡲 Preclear whole cell lysate (optional): To approximately
tube at 4° C. 1 ml of whole cell lysate or tissue extract in a 1.5 ml
microcentrifuge tube, add 40-50 µl of the suspended
䡲 To 1 ml of the above cell lysateor approximately (25% v/v) IP matrix supplied with each kit. Incubate
100–1000 µg of total cellular protein, add 10 µg of for 30 minutes at 4° C while rotating.
primary antibody agarose conjugate (i.e. 5 µl volume
of packed beads) and incubate at 4° C for 1 hour to
䡲 Pellet IP matrix via microcentrifugation at maximum
overnight with mixing. speed for 30 seconds at 4° C. Without disturbing pellet,
transfer desired supernatant (precleared cell lysate) to
䡲 Alternatively, if primary antibody agarose conjugate is a new microcentrifuge tube. Store precleared lysate
not available, incubate 1 ml cell lysate with 1–10 µl on ice and discard the pellet.
(i.e. 0.2–2 µg) primary antibody (optimal antibody
concentration should be determined by titration) for
䡲 Formation of the IP antibody-IP matrix complex: To a
1–2 hours at 4° C. Add 20 µl of appropriate agarose microcentrifuge tube, add 40-50 µl of suspended (25%
conjugate suspension (Protein A-Agarose, Protein G- v/v) IP matrix, 1–5 µg of IP antibody and 500 µl of PBS.
Agarose, Protein A/G-Agarose or Protein L-Agarose). Optimal antibody amount should be determined by
Cap tubes and incubate at 4° C on a rocker platform titration. Incubate at 4° C on a rotator for at least 1
or rotating device for 1 hour to overnight. hour. This step can be performed in parallel with the
above preclearing step or performed the day before
䡲 Collect pellet by centrifugation at 3,000 rpm (approxi- and allowed to incubate overnight at 4° C.
mately 1,000xg) for 30 seconds at 4° C. Carefully
NOTE: It is necessary that the species of the IP anti-
aspirate and discard supernatant.
body matches the species of the IP matrix included
䡲 Wash pellet 2–4 times with either RIPA buffer with each ExactaCruz™ kit. For example, when per-
(sc-24948) (more stringent) or PBS (less stringent), forming an IP with a mouse antibody, it must be incu-
each time repeating centrifugation step above. bated with the Mouse IP Matrix provided (sc-45040
or sc-45042).

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䡲 After incubation of the IP antibody with the species ExactaCruz™ E Alternate Protocol
specific IP matrix, pellet matrix via microcentrifugation 䡲 After transfer, block/wash membrane with TBST (10x
at maximum speed for 30 seconds at 4° C. Carefully
TBST: sc-24953) for 1 hour, changing TBST once half
aspirate and discard supernatant.
way through the incubation.
䡲 Wash pelleted matrix 2 times with 500 µl of PBS, each 䡲 Dilute WB antibody with ExactaCruz™ E Dilution
time repeating the above centrifugation and aspiration
Buffer (provided), add to membrane and incubate for
steps.
1–2 hours at room temperature.
䡲 Immunoprecipitation: After the final wash of the IP an- 䡲 After incubation, wash 3x with 1x TBST, 5 minutes per
tibody-IP matrix complex, transfer lysate (100-1000 µg
wash.
of total cellular protein) to the pelleted matrix and in-
cubate at 4° C on a rotator for 1 hour to overnight.
䡲 Dilute ExactaCruz E Western Blot Reagent (1:1000-
1:10000) with ExactaCruz E Dilution Buffer (provided),
䡲 After incubation of the matrix and lysate, microcen-
add to membrane and incubate 1–2 hours at room
trifuge at maximum speed for 30 seconds at 4° C to
temperature.
pellet. Aspirate and discard supernatant or alterna-
tively keep supernatant for another IP or testing via
䡲 Wash membrane 3x with TBST, 5 minutes per wash.
western blot. 䡲 Wash membrane once with 1x TBS (10x TBS: sc-24951)
䡲 Wash pelleted matrix 2–4 times with either RIPA Lysis for 5 minutes.
Buffer: sc-24948 (more stringent) or 1x PBS (less strin- 䡲 Incubate membrane in Western Blot Luminol Reagent:
gent), each time repeating the above centrifugation sc-2048 according to Luminol data sheet.
and aspiration steps.
5. IMMUNE COMPLEX PROTEIN KINASE
䡲 After final wash, aspirate and discard the supernatant ASSAYS
and resuspend pellet in 40-50 µl of reducing 2x Electro-
NOTE: For a listing of available cell culture products in-
phoresis Sample Buffer: sc-24945. Boil samples for
cluding classical and specialty media, sera and media
2–3 minutes. Note: The immunoprecipitated sample
additives, induction agents, antibiotics and attachment
must be completely reduced and denatured for Exacta-
agents, please see our catalog or visit our website at
Cruz™ to work properly.
www.scbt.com.
䡲 Perform a quick spin to pellet IP matrix and carefully 䡲 Remove medium from 100 mm cell culture plate
load supernatant onto gel. Continue with electrophor-
(80–90% confluent monolayer) and wash once with
esis as described under the Western (Immuno) Blotting
PBS.
procedure.
䡲 Add 1–3 ml ice cold RIPA buffer (sc-24948) to cell
䡲 At this stage it is essential that the immunoblotting
monolayer and incubate at 4° C for 10 minutes.
(primary) antibody matches the species specificity of
(Note: the use of RIPA buffer may not be optimal for
the HRP conjugated ExactaCruz™ detection reagent
some kinases. Composition of lysis buffer may need
which is unique for each kit. Detect the immunoblot-
to be optimized to maintain active kinase.)
ting (primary) antibody using the corresponding HRP
conjugated ExactaCruz™ reagent and Western Blot 䡲 Disrupt cells by repeated passage through a 21-gauge
Luminol Reagent: sc-2048. needle and transfer to microcentrifuge or 15 ml conical
centrifuge tube.
NOTE: When using sc-45042 ExactaCruz™ E, the alter-
nate immunoblotting protocol that is specific for this 䡲 Wash cell culture plate with addition of 1.0 ml ice cold
kit must be followed as described below in order to RIPA buffer, 0.5% Triton X-100 (Triton X-100: sc-29112)
generate desired results. and combine with original extract.
䡲 Pellet cellular debris at 10,000xg for 10 minutes at 4° C.

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Transfer supernatant to a new microcentrifuge or 15 ml may also choose to analyze immobilized peptides pre-
conical centrifuge tube at 4° C. pared by standard methods or offered commercially.
䡲 Transfer 1.0 ml cell extract (supernatant from above 6. IMMUNOPEROXIDASE CELL STAINING
step) to a 1.5 ml microcentrifuge tube. Add 1-10 µl
NOTE: For a listing of cover glasses and micro slides for
(i.e. 0.2–2 µg) primary antibody (optimal antibody
Immunohistochemistry, please see our catalog or visit our
concentration should be determined by titration) and
website at www.scbt.com.
incubate for 1 hour at 4° C.
䡲 Add 20 µl of appropriate agarose conjugate suspen- A. Tissue Culture Cells
sion (Protein A-Agarose, Protein G-Agarose, Protein NOTE: For a listing of available cell culture products in-
A/G-Agaroseor Protein L-Agarose). Cap tubes and in- cluding classical and specialty media, sera and media
cubate at 4° C on a rocker platform or rotating device additives, induction agents, antibiotics and attachment
for 1 hour to overnight. agents, please see our catalog or visit our website at
䡲 Collect immunoprecipitates by centrifugation at 2,500 www.scbt.com.
rpm (approximately 1,000xg) for 5 minutes at 4° C. 䡲 Grow cultured cells on sterile glass cover slips or
Carefully aspirate and discard supernatant. slides overnight at 37° C. Wash briefly with PBS and
䡲 Wash pellet four times with 1.0 ml RIPA buffer (more fix cells by one of the following procedures:
stringent) or PBS (less stringent), each time repeating 1) 5 minutes in -10° C methanol, air dry (recommended
centrifugation step above. method); or
䡲 Suspend pellet in 20 µl of the appropriate protein 2) 2 minutes in cold acetone, air dry; or
kinase assay buffer (e.g. 50 mM HEPES (HEPES:
sc-29097), 0.1 mM EDTA (EDTA: sc-29092), 0.01% 3) 10 minutes in 1% formalin in PBS (keep wet).
BRIJ® 35, 0.1 mg/ml BSA, 0.1% β-mercaptoethanol, 䡲 Wash in three changes of PBS.
0.15 M NaCl. Buffer composition will depend upon the Optional: Incubate for 5–10 minutes in 0.1-1% hydrogen
kinase under study. peroxide in PBS to quench endogenous peroxidase activ-
䡲 Add 10–1000 ng peptide substrate. Peptide substrate ity. Wash in PBS twice for 5 minutes each.
concentration should be determined empirically for
B. Frozen Tissue Sections
the substrate/enzyme/cell line used.
NOTE: For a listing of mounting media for Immunohisto-
䡲 Prepare 1 ml ATP mix: 930 µl appropriate protein kinase
assay buffer, 6 µl 50 mM ATP, pH 7.0, 20 µl 2.0 M chemistry including Clarion and Crystal Mounting Media,
see our catalog or visit our website at www.scbt.com.
MgCl2 and 44 µl [γ P]-ATP [10 mCi/ml]. Add 10 µl ATP
32

mix per sample and incubate for 20 minutes at 30° C. 䡲 Freeze tissue block in liquid nitrogen according to
Place on ice. standard procedures. Block may be stored at -70° C
䡲 Terminate the reaction by adding an equal volume of for up to 2 weeks before sectioning.
2x electrophoresis sample buffer (sc-24945) and boil 䡲 Clean glass slides with 95% ethanol, treat with sub-
samples for 2–3 minutes. After boiling, samples may bing solution and air dry. Or use pre-treated slides.
be centrifuged to pellet the agarose beads (optional); 䡲 Cut 4- to 10-micron thick sections. Adhere sections
the supernatant is analyzed. Analyze samples by SDS- to room temperature slides. Slides may be stored at
PAGE and autoradiography. Unused samples may be -70° C. Thaw slides at room temperature prior to fixing
stored at -20° C. Alternatively, labeled peptides can and staining.
be separated from unicorporated label by acid precipi-
tation followed by collection on a filter and radioactiv-
䡲 Fix slides in cold acetone for 10 minutes and keep re-
ity determined by scintillation counting. Researchers frigerated (or choose other fixation procedure). Wash
in three changes of PBS.

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Optional: Incubate for 5–10 minutes in 0.1–1% hydro- saponin in deionized H2O at room temperature.
gen peroxide in PBS to quench endogenous peroxidase Wash at least three times in PBS. Aspirate excess
activity. Wash in PBS twice for 5 minutes each. liquid from slides.
NOTE: For tissues containing high levels of endogenous Optional: Incubate for 5-10 minutes in 0.1-1% hydro-
biotin (which may result in higher background staining), gen peroxide in deionized H2O to quench endogenous
we recommend following the Formalin-Fixed, Paraffin- peroxidase activity. Wash in PBS twice for 5 minutes
Embedded Tissue Sections protocol, as endogenous each.
biotin is normally destroyed in paraffin-embedded
D. Immunoperoxidase Staining
tissue.
䡲 For immunoperoxidase staining of tissue sections, we
C. Formalin-Fixed, Paraffin-Embedded recommend the use of either the Santa Cruz Biotech-
Tissue Sections
nology, Inc. ABC Staining Systems or the ImmunoCruz™
䡲 Fix tissue sections in formalin and embed in paraffin Staining Systems. The ABC Staining Systems utilize
blocks according to standard procedures. preformed avidin-biotinylated horseradish peroxidase
䡲 Clean glass slides with 95% ethanol, treat with sub- complex as a detection reagent, whereas the Immuno-
bing solution and air dry. Or use pre-treated slides. Cruz™ Staining Systems utilize a streptavidin-horse-
radish peroxidase complex. The ImmunoCruz™ Staining
䡲 Cut 4-6 micron thick tissue sections and apply to
Systems include all secondary reagents in a pre-diluted,
slides. Deparaffinize in xylenes using three changes
ready to use format. Complete research protocols are
for 5 minutes each. Hydrate sections gradually through
included with all Staining Systems; brief protocols are
graded alcohols: wash in 100% ethanol twice for 10
given below.
minutes each, then 95% ethanol twice for 10 minutes
each. Wash in deionized H2O for 1 minute with stirring. 䡲 All steps are carried out at room temperature in a hu-
Aspirate excess liquid from slides. midified chamber. Allow all Staining System reagents
to reach room temperature prior to use. Tissue sections
Optional: Antigen unmasking may be performed at this
should not be allowed to dry out at any time during
point. Certain antigenic determinants are masked by
the procedure. Use suction to remove reagents after
formalin fixation and paraffin embedding and may be
each step, but avoid drying of specimens between
exposed by one of several methods:
steps. Use sufficient reagents to cover the specimens
1) Heat treatment (recommended method): Place (approximately 100 µl per slide is usually adequate).
slides in a container and cover with 10 mM sodium
ABC STAINING SYSTEMS
citrate buffer, pH 6.0; or with 50 mM glycine-HCl
buffer (glycine: sc-29096), pH 3.5, with 0.01% (w/v) 䡲 Incubate specimens for 1 hour in 1.5% normal block-
EDTA (EDTA: sc-29092). Heat at 95° C for 5 minutes. ing serum in PBS. Blocking serum ideally should be
Top off with fresh buffer and heat at 95° C for 5 derived from the same species in which the secondary
minutes (optimal incubation time may vary for each antibody is raised. Remove blocking serum from slides.
tissue type). Allow slides to cool in the buffer for 䡲 Incubate with primary antibody for 30 minutes at room
approximately 20 minutes. Wash in deionized H2O temperature or overnight at 4° C. Optimal antibody
three times for 2 minutes each. Aspirate excess concentration should be determined by titration; rec-
liquid from slides. ommended range is 0.5–5.0 µg/ml diluted in PBS with
2) Pepsin: Incubate sections for 10–20 minutes in 1.5% normal blocking serum. Wash with three changes
0.1% pepsin in 0.01 N HCl at room temperature. of PBS for 5 minutes each.
Wash slides several times in deionized H2O. Aspirate 䡲 Incubate for 30 minutes with biotin-conjugated second-
excess liquid from slides. ary antibody as provided or at approximately 1 µg/ml
3) Saponin: Incubate sections for 30 minutes in 0.05% diluted in PBS with 1.5% normal blocking serum. Wash

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with three changes of PBS for 5 minutes each. 䡲 Prepare slides as described above for immunoperoxi-
䡲 Incubate for 30 minutes with avidin biotin enzyme dase staining, omitting the final step involving treat-
reagent. Wash with three changes of PBS for 5 minutes ment of cells with H2O2.
each. 䡲 Use suction to remove reagents after each step, but
䡲 Incubate in peroxidase substrate as provided for 30 avoid drying of specimens between steps. Use suffi-
seconds to 10 minutes or until desired stain intensity cient reagent to cover the specimen (approximately
develops. Individual slides should be monitored to de- 100–500 µl per slide is adequate).
termine the proper development time. Wash sections 䡲 Incubate specimens with 10% normal blocking serum
in deionized H2O for 5 minutes. If desired, counterstain in PBS for 20 minutes to suppress non-specific binding
in Gill’s formulation #2 hematoxylin (sc-24973) for of IgG. Blocking serum ideally should be derived from
5–10 seconds. Immediately wash with several changes the same species in which the secondary antibody is
of deionized H2O. raised. Wash with PBS.
䡲 Dehydrate through alcohols and xylenes as follows: 䡲 Incubate with primary antibody for 60 minutes. Opti-
Soak in 95% ethanol twice for 10 seconds each, then mal antibody concentration should be determined by
100% ethanol twice for 10 seconds each, then xylenes titration; recommended range is 0.5–5.0 µg/ml in PBS
three times for 10 seconds each. Wipe off excess xy- with 1.5% normal blocking serum. Wash with three
lene. Immediately add 1-2 drops of permanent mount- changes of PBS for 5 minutes each.
ing medium (e.g. Clarion sc-24942), cover with a glass 䡲 Incubate for 45 minutes with either biotin-conjugated
coverslip (sc-24975) and observe by light microscopy. or fluorochrome-conjugated secondary antibody diluted
IMMUNOCRUZ™ STAINING SYSTEMS to 1–5 µg/ml in PBS with 1.5%-3% normal blocking
serum. Optimal antibody concentration should be
䡲 Incubate specimens for 20 minutes in 1–3 drops of
determined by titration. Wash with three changes of
serum block. Aspirate serum from slides.
PBS. If fluorochrome-conjugated secondary antibody
䡲 Dilute primary antibody in serum block to 0.5–5.0 µg/ is used, incubate in a dark chamber and omit the next
ml as determined by titration. Incubate for 2 hours. step.
Rinse with PBS then wash in PBS twice for 2 minutes
䡲 Incubate with streptavidin-fluorescein for 15 minutes
each on a stir plate. Aspirate excess liquid from slides.
in a dark chamber. Optimal streptavidin conjugate
䡲 Incubate for 30 minutes in 1–3 drops of biotinylated concentration for a given application should be deter-
secondary antibody. Wash as above. mined by titration; recommended range is 10–20 µg/
䡲 Incubate for 30 minutes in 1–3 drops of HRP-strepta- ml in PBS. Wash extensively with PBS.
vidin complex. Wash as above. 䡲 Mount coverslip with aqueous mounting medium or
䡲 Add 1-3 drops HRP substrate mixture. Develop for 30 90% glycerol in PBS.
seconds to 10 minutes or until desired stain intensity 䡲 Examine using a fluorescence microscope with appro-
develops. Rinse with deionized H2O and transfer to a priate filters. Store slides in a dark location at room
deionized H2O wash for 2 minutes on a stir plate. temperature (UltraCruz™ Mounting Medium: sc-24941
䡲 Counterstain, dehydrate and mount slides as described or at 4° C (glycerol/PBS mount).
under ABC Staining Systems.
8. FLOW CYTOMETRY
7. IMMUNOFLUORESCENCE CELL STAINING
A. Sample Preparation
NOTE: For a listing of cover glasses and micro slides for
Prepare cells according to cell type.
Immunohistochemistry, please see our catalog or visit our
website at www.scbt.com.

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BLOOD (HUMAN, MOUSE OR RAT) 䡲 Add approximately 5 ml of 0.2% EDTA (in PBS) to
䡲 For each 1 ml of blood, add 14 ml of room temperature plate. Using a Trypsin/EDTA solution in the place of
FCM Lysing solution (sc-3621) to lyse the red blood 0.2% EDTA may compromise any cell surface staining.
cells. The cells will not lyse correctly if the solution is 䡲 Wait for cells to “round up.” Placing the cells in an in-
cold. cubator may speed up this process. Check the plate(s)
䡲 Incubate for 5 minutes at room temperature on a rota- every 5 minutes.
tor. Do not exceed 5 minutes, as the white blood cells 䡲 Add approximately 5 ml of media to neutralize EDTA.
will begin to lyse beyond 5 minutes. 䡲 Pipette off cells, rinsing plate to ensure maximum re-
䡲 Centrifuge for 5 minutes at 1000 RPM for human blood, covery. Take a small sample to perform a cell count.
2000 RPM for mouse or rat blood. 䡲 Centrifuge for 5 minutes at 1000 RPM.
䡲 Carefully aspirate supernatant, then resuspend pellet 䡲 Aspirate supernatant.
in approximately 50 ml cold 1X PBS. Take a small
sample to perform a cell count. B. Cell Stimulation
䡲 Centrifuge for 5 minutes at 1000 RPM for human blood, Stimulate cells as necessary.
2000 RPM for mouse or rat blood. C. Stain Preparation
䡲 Aspirate supernatant.
Fix cells or prepare live cells for staining.
MOUSE SPLEEN OR OTHER TISSUE
NOTE: It is very important to block Fc receptors for cer-
䡲 Harvest organ or tissue and prepare single cell sus- tain cell types including, but not limited to, mouse and
pension. rat blood, mouse spleen, mouse bone marrow, etc. For
䡲 Pass cell suspension through a 70 µM cell strainer. mouse or rat tissues, use sc-18867 L.
䡲 Centrifuge for 5 minutes at 1000 RPM. LIVE STAINING
䡲 Discard supernatant and add 5 ml of room temperature 䡲 Once supernatant is aspirated from cell preparation,
FCM Lysing solution (sc-3621). resuspend pellet in enough 1X PBS to have a final cell
concentration of 10 million cells/ml.
䡲 Incubate for 2-3 minutes at room temperature, allow-
ing larger pieces to fall to the bottom of the tube. 䡲 Block by incubating the cell suspension with 1 mg of
sc-18867 L per 1 ml of cell suspension for 10 minutes.
䡲 Carefully pipette the suspension out and deposit into
Do not rinse. Proceed with staining.
a clean tube. Take a small sample to perform a cell
count. FIXED AND PERMEABILIZED CELLS FOR INTRACELLULAR
STAINING
䡲 Centrifuge for 5 minutes at 1000 RPM.
䡲 Once supernatant is aspirated from cell preparation,
䡲 Aspirate supernatant. resuspend pellet in enough 1X PBS to have a final cell
SUSPENSION CELL LINE concentration of 10 million cells/ml.
䡲 Pipette off cells, rinsing plate to ensure maximum re- 䡲 Block by incubating the cell suspension with 1 µg of
covery. Take a small sample to perform a cell count. sc-18867 L per 1 ml of cell suspension for 10 minutes.
䡲 Centrifuge for 5 minutes at 1000 RPM. 䡲 Resuspend pellet in approximately 50 ml 1X PBS to
䡲 Aspirate supernatant. wash away any excess blocking antibody.

MONOCLONAL/ADHERENT CELL LINE


䡲 Centrifuge for 5 minutes at 1000 RPM.
䡲 Once supernatant is aspirated from cell preparation,
䡲 Vacuum off media. Rinse plate once with 1X PBS.
resuspend pellet in FCM Fixation Buffer (sc-3622).
Vacuum off PBS.
Use mL per million cells.

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䡲 Incubate for 30 minutes at room temperature on a DIRECT STAINING
rotator. (WITH FLUOROCHROME -CONJUGATED ANTIBODIES)

䡲 Centrifuge for 5 minutes at 1500-2000 RPM. Cells get 䡲 Label tubes.


more buoyant after fixation. If pellet is too small, spin 䡲 Add 20 µl of fluorochrome-conjugated antibodies to
again at a higher RPM, but do not exceed 3000 RPM. tubes.
䡲 Pour off supernatant. Cells may be lost if aspirating 䡲 Add 100 µl of the prepared cell suspension (equal to
from this point on, so always decant. Use a quick 1 million cells) to each tube.
motion and don’t allow the supernatant to wash back 䡲 Vortex and incubate for 15-30 minutes in a covered ice
and forth over the cells.
bucket.
䡲 Resuspend pellet in approximately 50 ml 1X PBS to 䡲 To wash off excess antibody following staining, add
wash away any excess Fixation Buffer.
1.5–2 ml of 1X PBS to each tube.
䡲 Centrifuge for 5 minutes at 1500–2000 RPM. 䡲 Centrifuge in tabletop microfuge for 5 minutes at 2000
䡲 Decant supernatant. At this point, cells can be resus- RPM. This speed should be increased to 3000 or 4000
pended in a small amount of PBS and stored for up to RPM for intracellular staining.
one month at 4° C. To permeabilize at this time, pro- 䡲 Aspirate supernatant, being careful not to disturb pellet.
ceed to next step.
䡲 Resuspend pellets in 500 µl of 1% paraformaldehyde.
NOTE: You should only proceed with permeabilization
Tubes can be stored in the dark for 24 hours (maximum
if you can stain immediately afterwards.
for intracellular staining) to 1 week (maximum for sur-
䡲 If cells have been stored in PBS, centrifuge for 5 min- face staining).
utes at 1500-2000 RPM and decant supernatant.
INDIRECT STAINING
䡲 Break up cell pellet and dropwise add the same amount (WITH FLUOROCHROME -UNCONJUGATED PRIMARY
of COLD (stored at -20° C) FCM Permeabilization Buffer, ANTIBODIES AND FLUOROCHROME-CONJUGATED
SECONDARY ANTIBODIES)
sc-3623 at 1 ml per 1 million cells. Vortex while adding.
䡲 Incubate for 5 minutes only at RT on a rotator.
䡲 Label tubes.
䡲 Immediately centrifuge for 5 minutes at 2000–2500
䡲 Add unconjugated primary antibodies to tubes. Use
RPM. Cells are more buoyant after permeabilization approximately 1 µg per tube.
and much care must be excercised to maintain volume 䡲 Add 100 µl of the prepared cell suspension (equal to
of cells. 1 million cells) to each tube.
NOTE: Important: If a pellet is not recovered at this 䡲 Vortex and incubate for 15-30 min in a covered ice
step, be sure to spin again and try to recover more bucket.
cells. 䡲 To wash off excess antibody following staining, add
䡲 Decant supernatant and add approximately 50 ml 1X 1.5–2 ml of 1X PBS to each tube.
PBS to wash away any excess Permeabilization Buffer. 䡲 Centrifuge in tabletop microfuge for 5 minutes at 2000
䡲 Centrifuge for 5 minuntes at 2000–2500 RPM. RPM (or 3000–4000 RPM for intracellular staining).
䡲 Decant supernatant and resuspend pellet in enough 䡲 Aspirate supernatant, being careful not to disturb pellet.
FCM Wash Buffer, sc-3624, for a final cell concentra- 䡲 Add 100 ml of 1X PBS to each tube. Add fluoro-
tion of 10 million cells/ml. In the staining steps, use chrome-conjugated secondary antibodies to tubes.
FCM Wash Buffer in place of 1X PBS. Use 0.5–1 µg of antibody.
D. Staining 䡲 Vortex and incubate for 15–30 minutes in a covered ice
Follow protocol for direct or indirect staining. bucket.

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䡲 To wash off excess antibody following staining, add 䡲 Remove liquid in wells. Wash three times with PBS
1.5–2 ml of 1X PBS to each tube. containing 0.05% Tween-20 and dry plate.
䡲 Centrifuge in tabletop microfuge for 5 minutes at 2000 䡲 Wash wells once with diethanolamine buffer (10 mM
RPM (or 3000–4000 RPM for intracellular staining). diethanolamine, 0.5 mM MgCl2, pH 9.5) and remove
䡲 Aspirate supernatant, being careful not to disturb pellet. liquid.
䡲 Resuspend pellets in 500 µl of 1% paraformaldehyde.
䡲 Dilute substrate (PNPP, sc-3720) in diethanolamine
Tubes can be stored in the dark for 24 hours (maximum buffer to a final concentration of 1 mg/ml. Add 50 µl/
for intracellular staining) to 1 week (maximum for sur- well. Allow to develop for 10–20 minutes or until
face staining). positive control reaches an OD 405/490 of about 1.0.
Stop reaction by adding 50 µl of 0.1 M EDTA (EDTA:
E. Acquire sc-29092), pH 7.5. Read plates on microtiter plate
Acquire within 24 hours. reader at OD 405/490.

9. ELISA ASSAYS 10. TRANSCRUZ™GEL SUPERSHIFT ASSAYS


䡲 Coat microtiter plates with target protein diluted in 䡲 Label oligonucleotide probe with [γ P]-ATP to 50,000
32

50 mM carbonate buffer at pH 9.0. Optimal concen cpm/ng by using polynucleotide kinase (for a listing of
trations should be determined by titration, but for these reagents and more, please see our catalog or
purified antigens 50 µl per well at 1 µg/ml is usually visit our website at www.scbt.com.).
sufficient. Incubate overnight at 4° C covered with 䡲 Prepare gel shift reaction buffer as follows: 10 mM
parafilm. Tris (Tris: sc-3715), pH 7.5, 50 mM NaCl, 1 mM dithio-
䡲 Remove antigen solution. Add 200 µl/well of blocking threitol (DTT: sc-29089), 1 mM EDTA (EDTA: sc-29092),
buffer (PBS containing 1% BSA and 0.02% azide) to 5% glycerol (glycerol: sc-29095).
block non-specific protein binding. Incubate for 1–2 䡲 Prepare 20 µl reaction mixture containing 3–10 µg
hours at room temperatureor overnight at 4° C. nuclear extract and 1 µg poly dI-dC in gel shift reac-
䡲 Remove blocking buffer. Wash once with PBS with tion buffer. Add 0.5 ng labeled oligonucleotide probe
0.02% azide. Damp strip wells or plates are usually and incubate for 20 minutes at room temperature.
stable in resealable plastic storage bags for 4 weeks This constitutes the control sample for detection of
at 4° C. Before using, remove excess liquid. DNA-protein complexes.
䡲 Add test antibody samples and controls at 50 µl/well 䡲 To detect an antibody supershift or block of the DNA-
diluted in blocking buffer. Antibodies may be serially protein complex, prepare reaction mixture as described
diluted for determining titer or diluted to previously above, also adding 1–2 µl of the appropriate Trans-
determined working concentration for screening as- Cruz™ Gel Supershift antibody per 20 µl of reaction
says or antigen quantitation. Incubate 1 hour at room volume. Antibody is normally added subsequent to ad-
temperature. dition of labeled oligonucleotide probe, but result may
be improved by adding antibody prior to probe. Incubate
䡲 Wash plates three times with PBS containing 0.05%
at 4° C for 1 hour to overnightor at room temperature
Tween-20 (Tween-20: sc-29113), removing excess
for 15–45 minutes.
liquid as above.
䡲 Resolve DNA-protein complexes by electrophoresis
䡲 Add 50 µl/well of alkaline phosphatase-conjugated
(25–35 ma) through a 4% polyacrylamide gel containing
secondary antibody diluted to 1:100–1:1000 in block-
50 mM Tris, pH 7.5, 0.38 M glycine (glycine: sc-29096)
ing buffer. Optimal antibody concentration is deter-
and 2 mM EDTA. Dry the gel and visualize by auto-
mined by titration. Incubate 1 hour at room
radiography.
temperature.

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11. PEPTIDE NEUTRALIZATION fuge tube for the sonication step.
䡲 Blocking (neutralizing) peptides are available as neg- 䡲 Sonication conditions should be optimized since re-
ative controls for all Santa Cruz Biotechnology, Inc. sults may vary using different sonifiers. The following
affinity-purified rabbit and goat polyclonal antibodies conditions were established by using a Sonics VC130
and monoclonal antibodies raised against peptide with a 3 mm tip probe.
antigens. Antibody binding to antigen may be blocked/ 䡲 Sonicate on ice at power output setting = 5-6, continu-
competed by pre-absorption with the blocking peptide. ous mode, 4 times at 30 second intervals.
䡲 Determine the highest antibody dilution at which a 䡲 Centrifuge extract for 15 minutes, 10,000 rpm at 4° C
consistently positive result is achieved for the desired and save supernatant (chromatin).
test. For example, H-Ras (259) is recommended for im- 䡲 Determine protein concentration of supernatant.
munoprecipitation at 1 µg/ml but is positive at a dilution
of 50 ng/ml. 䡲 For the IP step we recommend using 100–500 µg
protein and 0.1-1 µl TransCruz™ reagent (0.2-2 µg).
䡲 For blocking/competition, combine antibody (at a con-
centration determined by the aforementioned method) NOTE: Investigators may wish to consider using the
with a five-fold (by weight) excess of blocking peptide primary antibody conjugated to sepharose or magnetic
in a small volume (500 µl) of PBS. Incubate for up to beads as an alternative to using secondary immuno-
2 hours at room temperature or overnight at 4° C. precipitation reagents (e.g. Protein A-Agarose) as de-
scribed here. Combining primary antibodies directed
䡲 Following blocking/competition, dilute antibody/pep-
to different epitopes of the same protein may be ad-
tide mixture into appropriate blocking buffer and pro-
vantageous in some cases.
ceed with the desired research application.
䡲 Preclear the chromatin solution by adding 50 µl Pro-
12. CHROMATIN IMMUNOPRECIPITATION tein A/G PLUS-Agarose (sc-2003) and incubate for 30
(ChIP) ASSAYS
minutes at 4º C. Centrifuge at full speed for 5 minutes
NOTE: ChIP protocols vary widely. The following protocol at 4º C.
should be suitable for most experiments. 䡲 Add primary antibody to the supernatant and incubate
䡲 Wash cells twice with PBS at room temperature, re- overnight at 4° C.
suspending to approximately 5x10 cells/ml (approxi-
5
䡲 Add 50 µl Protein A/G PLUS-Agarose (sc-2003) and
mately 2x10 cells total). Add formaldehyde to a final
7

incubate for 2 hrs at 4º C.


concentration of 1% and incubate at room temperature
for 10 minutes.
䡲 Harvest beads by centrifugations at 12,000 rpm for 20
seconds and place tube in ice.
䡲 Terminate cross-linking reactions by adding glycine to
a final concentration of 0.125 M.
䡲 Wash beads twice with 1 ml Lysis Buffer High Salt
(sc-45001).
䡲 Pellet cells (2,000 rpm, 5 minutes) and wash once with
ice cold PBS.
䡲 Wash pellet four times with Wash Buffer (sc-45002).
䡲 Resuspend cells in 6 ml Lysis Buffer (sc-45000) by
䡲 Resuspend beads in 400 µl Elution Buffer (sc-45003).
mixing gently. 䡲 Reverse cross-links by incubating tube in a 67º C water
䡲 Collect crude nuclear extract by microcentrifugation at bath, mixing occasionally over two hours. Remove
2,000 rpm, 5 minutes.
beads by centrifugation and continue incubating super-
䡲 Wash again with PBS. Pellet may be frozen or process- natant at 67º C overnight.
ing may be continued as follows:
䡲 Centrifuge for 3 minutes at 10,000 to remove any
䡲 Resuspend pellet in approximately1.9 ml Lysis Buffer residual beads and save supernatant.
High Salt (sc-45001) and transfer to 2 ml microcentri-

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䡲 To isolate DNA, extract supernatant once with 500 µl NOTE: Do not add serum and antibiotics to the siRNA
phenol/chloroform/isoamyl alcohol (25:24:1), vortex Transfection Medium: sc-36868.
thoroughly and separate phases by centrifuging tube NOTE: Optimal siRNA amount used for transfection
for 3 minutes at 14,000 rpm. may vary for each target protein and should be deter-
䡲 Save the aqueous phase, back extract the organic mined experimentally.
phase once with 100 µl 10 mM Tris, 1 mM EDTA, NOTE: If a lower siRNA concentration is desired, dilute
pH 8.1 (TE) and pool aqueous phases. siRNA appropriately with siRNA Dilution Buffer:
䡲 Extract pooled aqueous phase with 600 µl chloro- sc-29527.
form/isoamyl alcohol. NOTE: Although highly efficient in a variety of cell
䡲 DNA may be concentrated by using commercially lines, siRNA Transfection Reagent: sc-29528 may not
available kits. be suitable for use with all cell lines.
13. siRNA MEDIATED INHIBITION OF GENE 䡲 Add the siRNA duplex solution (Solution A) directly
EXPRESSION to the dilute Transfection Reagent (Solution B) using a
pipette. Mix gently by pipetting the solution and incu-
NOTE: Santa Cruz Biotechnology, Inc. offers siRNA prod-
bate the mixture 15–45 minutes at room temperature.
ucts for every human and mouse protein for which we
have a corresponding antibody. Most are listed through- 䡲 Wash the cells once with 2 ml of siRNA Transfection
out the catalog in the appropriate sections. For a com- Medium: sc-36868 Aspirate the medium and proceed
plete list of all of the siRNA products we offer, please immediately to the next step.
see our web site at www.scbt.com. 䡲 For each transfection, add 0.8 ml siRNA Transfection
䡲 In a six well tissue culture plate, seed 2 x 10 cells per 5 Medium (Solution A + Solution B) to each tube con-
well in 2 ml antibiotic-free normal growth medium taining the siRNA: Transfection reagent mixture. Mix
supplemented with FBS. gently and overlay the mixture onto the washed cells.
NOTE: This protocol is recommended for a well from 䡲 Incubate the cells 5–7 hours at 37° C in a CO2 incubator.
a 6 well tissue culture plate. Adjust cell and reagent NOTE: Longer transfection times may be desirable
amounts proportionately for wells or dishes of differ- depending on the cell line. However prolonged serum
ent sizes. starvation may result in unwanted cell detachment or
䡲 Incubate the cells at 37° C in a CO2 incubator until death.
the cells are 60 - 80% confluent. This typically takes NOTE: Fluorescein Conjugated Control siRNA should
18–24 hours. only be incubated for a total 5-7 hours at 37° C in a
NOTE: Healthy and subconfluent cells are required for CO2 incubator. At the end of incubation they are ready
successful transfection experiments. It is recommended to be assayed by fluorescent microscopy.
to ensure cell viability one day prior to transfection. 䡲 Add 1 ml of normal growth medium containing 2 times
䡲 Prepare the following solutions: the normal serum and antibiotics concentration (2x
normal growth medium) without removing the trans-
Solution A: For each transfection, dilute 2–8 µl of
fection mixture. If toxicity is a problem, remove the
siRNA duplex (i.e. 0.25–1 µg or 20-80 pmols siRNA)
transfection mixture and replace with 1x normal
into 100 µl siRNA Transfection Medium: sc-36868.
growth medium.
Solution B: For each transfection, dilute 2–8 µl of
䡲 Incubate the cells for an additional 18–24 hours.
siRNA Transfection Reagent: sc-29528 into 100 µl
siRNA Transfection Medium: sc-36868. Peak activity 䡲 Aspirate the medium and replace with fresh 1x normal
should be at about 6 µl siRNA Transfection Reagent. growth medium.

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䡲 Assay the cells using the appropriate protocol 24–72 䡲 Incubate at 42° C for 2 minutes to anneal primer and
hours after the addition of fresh medium in the step template.
above. 䡲 Add 1 µl reverse transcriptase (200 units) and incubate
NOTE: Controls should always be included in siRNA at 42° C for 50 minutes to extend the primer and then
experiments. Use either Control siRNAs or Control terminate the reaction by incubating at 70° C for 15
siRNA (Fluorescein Conjugates) (see table above). Each minutes.
contain a scrambled sequence that will not lead to the NOTE: As an optional step add 1 µl RNase H (2 unit/µl)
specific degradation of any known cellular mRNA. and incubate at 37° C for 20 minutes.
NOTE: For Western blot analysis prepare cell lysate
2. First PCR Reaction
as follows: Wash cells once with PBS. Lyse cells in
300 µl 1x electrophoresis sample buffer (sc-24945: 䡲 Prepare a solution containing:
Electrophoresis Sample Buffer, 2X) by gently rocking 5 µl 10x PCR buffer (with or without* MgCl2)
the 6 well plate or by pipetting up and down. Sonicate 5 µl 25 mM MgCl2
the lysate on ice if necessary.
1 µl 10 mM dNTP
NOTE: For RT-PCR analysis isolate RNA using the 1 µl primer pair A
method described by Chomczynski and Sacchi (1987. 1 µl Taq DNA polymerase
Anal. Biochem. 162:156-159. Single-step method of 2 µl cDNA and add water to 50 µl
RNA isolation by acid guanidinium thiocyanate-phenol- * It may be necessary to vary the MgCl2 concentration, 2.5 mM final
chloroform extraction. Chomczynski P, Sacchi N.) or a concentration recommended.)
commercially available RNA isolation kit. 䡲 Incubate at 94° C for 2 minutes to denature the cDNA.
14. SEMI-QUANTITATIVE NESTED RT-PCR 䡲 Perform 15–40 PCR cycles. Annealing and extension
NOTE: Tm values for PCR primers offered by Santa Cruz conditions are primer and template dependent and
Biotechnology, Inc. range between 55-60 C (19-21 nt, must be determined empirically for each template-
GC% ~55%). The A and B nested primer sets share primer pair.
similar base pair length, GC% and Tm values. 3. Second PCR Reaction
NOTE: Nested PCR utilizes two pairs of PCR primers for a 䡲 Prepare a solution containing:
single locus. The first primer pair A set amplifies within
the locus. The second primer pair B set (nested primers) 5 µl 10x PCR buffer (with or without* MgCl2)
then binds within the “A” amplicon to produce a second 1 µl 10 mM dNTP
nested “B” amplicon. 1 µl primer pair B
1 µl Taq DNA polymerase
1. cDNA Synthesis 1-5 µl first PCR product and add water to 50 µl
䡲 Prepare a solution containing: * It may be necessary to vary the MgCl2 concentration, 2.5 mM final
concentration recommended.)
1 µl oligo (dT)12-18 (500 µg/ml)
1 ng-5 µg total RNA
䡲 Incubate at 94° C for 2 minutes to denature the cDNA.
1 µl 10 mM dNTPs 䡲 Perform 15–40 PCR cycles. Annealing and extension
and add RNase-free water to a final volume of 12 µl conditions are primer and template dependent and
must be determined empirically for each template-
䡲 Incubate at 70° C for 5 minutes to minimize RNA
primer pair.
secondary structure, quick chill on ice and then add:
4 µl 5x reverse transcriptase buffer
䡲 PCR products are separated on agarose gels and
visualized by ethidium bromide staining.
2 µl 0.1 M DTT
1 µl RNase inhibitor

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15. shRNA PLASMID DNA MEDIATED NOTE: Although highly efficient in a variety of cell
INHIBITION OF GENE EXPRESSION lines, shRNA Plasmid Transfection Reagent: sc-
䡲 In a six well tissue culture plate, grow cells to a 108061 may not be suitable for use with all cell
50-70% confluency in antibiotic-free normal growth lines.
medium supplemented with FBS. 䡲 Add the shRNA Plasmid DNA solution (Solution A)
NOTE: This protocol is recommended for a well from directly to the dilute shRNA Plasmid Transfection
a 6 well tissue culture plate. Adjust cell and reagent Reagent (Solution B) using a pipette. Mix gently by
amounts proportionately for wells or dishes of differ- pipetting the solution up and down and incubate the
ent sizes. mixture 15-45 minutes at room temperature.
NOTE: Healthy and subconfluent cells are required for 䡲 Wash the cells twice with 2 ml of shRNA Transfection
successful transfection experiments. It is recom- Medium: sc-108062 Aspirate the medium and proceed
mended to ensure cell viability one day prior to immediately to the next step.
transfection. NOTE: Do not use PBS as the residual phosphate may
䡲 Prepare the following solutions: compete with DNA and bind the shRNA Plasmid
Transfection Reagent, thereby reducing the transfection
NOTE: The optimal shRNA Plasmid DNA:shRNA Plas- efficiency.
mid Transfection Reagent ratio should be determined
experimentally beginning with 1 µg of shRNA Plasmid 䡲 For each transfection, add 0.8 ml shRNA Plasmid
DNA and between 1.0 and 6.0 µl of shRNA Plasmid Transfection Medium to well.
Transfection Reagent as outlined below. Once the 䡲 Add the 200 µl shRNA Plasmid DNA/shRNA Plasmid
optimal shRNA Plasmid DNA:shRNA Plasmid Trans- Transfection Reagent Complex (Solution A + Solution
fection Reagent ratio has been identified for a given B) dropwise to well, covering the entire layer.
cell type, the appropriate amount of shRNA Plasmid 䡲 Gently mix by swirling the plate to ensure that the en-
DNA/shRNA Plasmid Transfection Reagent complex tire cell layer is immersed in solution.
used per well should be tested to determine which
amount provides the highest level of transfection effi-
䡲 Incubate the cells 5-7 hours at 37° C in a CO2 incuba-
ciency. For example, if the optimal shRNA Plasmid tor or under conditions normally used to culture the
DNA:shRNA Plasmid Transfection Reagent ratio is cells. Longer transfection times may be desirable de-
1 µg:1 µl, then amounts ranging from 0.5 µg/0.5 µl pending on the cell line.
to 2.0 µg/2.0 µl should be tested. 䡲 Following incubation, add 1 ml of normal growth
medium containing 2 times the normal serum and an-
Solution A: For each transfection, dilute 10 µl of resus-
tibiotics concentration (2x normal growth medium).
pended shRNA Plasmid DNA (i.e. 1 µg shRNA Plasmid
DNA) into 90 µl shRNA Plasmid Transfection Medium: 䡲 Incubate the cells for an additional 18-24 hours under
sc-108062. conditions normally used to culture the cells.
Solution B: For each transfection, dilute 1-6 µl of OPTIONAL: For transient transfection, aspirate media
shRNA Plasmid Transfection Reagent: sc-108061 and replace with fresh1x normal growth medium.
with enough shRNA Plasmid Transfection Medium: Assay the cells using the appropriate protocol 24-72
sc-108062 to bring final volume to 100 µl. hours after the addition of fresh medium in the previ-
ous step.
NOTE: Do not add antibiotics to the shRNA Plasmid
Transfection Medium: sc-108062. For selection of stably transfected cells, proceed with
puromycin selection as follows:
NOTE: Optimal results may be achieved by using sili-
conized microcentrifuge tubes.

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NOTE: The working puromycin concentration for mam- should be adjusted depending on the growth area of
malian cell lines ranges from 1-10 µg/ml. Prior to the well or plate.
using the puromycin antibiotic (sc-108071), titrate the DAY 2
selection agent to determine the optimal concentra-
tion for target cell line. Use the lowest concentration
䡲 Prepare a mixture of complete medium with Poly-
that kills 100% of non-transfected cells in 3-5 days brene® (sc-134220) at a final concentration of 5 µg/
from the start of puromycin selection. ml.
䡲 Remove media from plate wells and replace with 1 ml
48 hours post-transfection, aspirate the medium and
of this Polybrene/ media mixture per well (for 12-well
replace with fresh medium containing puromycin at
plate).
the appropriate concentration.
NOTE: Polybrene is a polycation that neutralizes
Approximately every 2-3 days, aspirate and replace
charge interactions to increase binding between the
with freshly prepared selective media.
pseudoviral capsid and the cellular membrane. The
NOTE: Controls should always be included in shRNA optimal concentration of Polybrene depends on cell
experiments. Control shRNAs are available as 20 µg. type and may need to be empirically determined (usu-
Each encode a scrambled shRNA sequence that will ally in the range of 2-10 µg/ml). Excessive exposure to
not lead to the specific degradation of any known Polybrene (> 12 hr) can be toxic to some cells.
cellular mRNA. Control shRNA Plasmids include: 䡲 Thaw lentiviral particles at room temperature and mix
sc-108060, sc-108065 and sc-108066. gently before use.
NOTE: For Western blot analysis prepare cell lysate as 䡲 Infect cells by adding the shRNA Lentiviral Particles to
follows: Wash cells once with PBS. Lyse cells in 300 µl the culture.
1x Electrophoresis Sample Buffer (sc-24945) by gently 䡲 Swirl the plate gently to mix and incubate overnight.
rocking the 6 well plate or by pipetting up and down.
The amount of viral particles to use varies greatly de-
Sonicate the lysate on ice if necessary.
pending on the characteristics of the cell line used.
NOTE: For RT-PCR analysis isolate RNA using the
NOTE: Keep thawed shRNA Lentiviral Particles on ice.
method described by P. Chomczynski and N. Sacchi
Repeated freeze-thaw cycles and prolonged exposure
(1987. Single-step method of RNA isolation by acid
of the particles to ambient temperatures may result in
guanidinium thiocyanate-phenol-chloroform extrac-
decreased viral titers.
tion. Anal. Biochem. 162: 156-159) or a commercially
available RNA isolation kit. NOTE: When transducing a shRNA lentiviral construct
into a cell for the first time we suggest using several
16: shRNA LENTIVIRAL PARTICLES amounts of shRNA lentiviral particle stock. In addition,
TRANSDUCTION we recommend to include one well with cells trans-
DAY 1 duced with Control shRNA Lentiviral Particles (sc-
䡲 Plate target cells in a 12-well plate 24 hours prior to 108080).
viral infection. NOTE: Use copGFP Control Lentiviral Particles (sc-
䡲 Add 1 ml of complete optimal medium (with serum 108084) for measuring transduction efficiency.
and antibiotics) and incubate cells overnight. The cells DAY 3
should be approximately 50% confluent on the day of 䡲 Remove the culture medium and replace with 1 ml of
infection and (Day 2). complete medium (without Polybrene).
NOTE: It is possible to use other plate formats for 䡲 Incubate the cells overnight.
transduction as well. In this case, the amount of cells

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DAY 4 inactivate after transduction and integration of shRNA
䡲 To select stable clones expressing the shRNA, split constructs into genomic DNA of target cells.
cells 1:3 to 1:5, depending on the cell type, and con- 17. GENERAL SOLUTIONS
tinue incubating for 24-48 hours in complete medium.
NOTE: For a listing of reagents required to prepare the
DAY 5-6 and forward following solutions, please see our catalog or visit our
䡲 Select stable clones expressing the shRNA via website at www.scbt.com.
Puromycin dihydrochloride (sc-108071) selection. 䡲 Blotto A (for general use): 1x TBS, 5% milk, 0.05%
䡲 For puromycin selection, use an amount sufficient to Tween-20. Available pre-made (sc-2333).
kill the non-transduced cells. Puromycin concentrations 䡲 Blotto B (for use with anti-phosphotyrosine antibodies):
ranging from 2 to 10 &mico;g/ml are usually sufficient,
1x TBS, 1% milk, 1% BSA, 0.05% Tween-20. In some
but a puromycin tritation is recommended when using
cases, milk may be left out entirely, but this will result
a new cell line.
in somewhat higher backgrounds. Available pre-made
䡲 Replace medium with fresh puromycin-containing (sc-2335). For all phospho-specific antibodies, add
medium every 3-4 days, until resistant colonies can be 0.01% (v/v) of each Phosphatase Inhibitor Cocktail A
identified. Pick several colonies, expand them and and B (sc-45044 and sc-45045) to inhibit phosphatase
assay them for stable shRNA expression. activity.
NOTE: Resulting puromycin-resistant clones may have 䡲 Diaminobenzidine tetrahydro-chloride (DAB): Dissolve
varying levels of shRNA expression due to the random 5 mg DAB in 100 ml 100 mM Tris-HCl, pH 7.6 and add
integration of the lentiviral construct into the genome 0.1 ml 0.3% hydrogen peroxide. Prepare fresh DAB
of the cell. solution daily.
NOTE: For shRNA expression analysis by Western 䡲 Electrophoresis sample buffer (2X): 100mM 2-(N-Mor-
Blot, prepare cell lysate as follows: pholino)- ethanesulfonic acid(MES), 10 mM Na EDTA,
䡲 Wash cells once with PBS. 15% glycerol, 1.5 %SDS, 0.3% Triton X, 25mM TCEP-
HCL, 7.5 mM DTT, 0.0025% Bromophenol Blue. Avail-
䡲 Lyse cells in 100 µl of a 1:1 mixture of 2x Electropho-
able pre-made (sc-24945).
resis Sample Buffer (sc-24945) and RIPA Lysis Buffer
(sc-24948) by gently rocking the 12-well plate or by 䡲 Phosphate buffered saline (1x PBS): 9.1 mM dibasic
pipetting up and down. sodium phosphate, 1.7 mM monobasic sodium phos-
phate and 150 mM NaCl. Adjust pH to 7.4 with NaOH.
䡲 Sonicate the lysate on ice if necessary.
Available pre-made in liquid (sc-24946) and powder
NOTE: For shRNA expression analysis by RT-PCR, iso- (sc-24947) forms.
late RNA using the method described by P. Chomczyn-
䡲 RIPA buffer: 1x PBS, 1% Nonidet P-40 or Igepal CA-630,
ski and N. Sacchi (1987. Single-step method of RNA
0.5% sodium deoxycholate, 0.1% SDS. This may be
isolation by acid guanidinium thiocyanate-phenol-
made in large volumes. Add protease inhibitors at
chloroform extraction. Anal. Biochem. 162: 156-159)
time of use from the following stock solutions. Also
or a commercially available RNA isolation kit.
available pre-made (sc-24948).
BIO SAFETY
1) 10 mg/ml PMSF (sc-3597) in isopropanol (add at
Lentiviral particles can be employed in standard Bio- 10 µl/ml RIPA)
safety Level 2 tissue culture facilities (and should be
2) Aprotinin (sc-3595) (add at 50 KIU/ml RIPA)
treated with the same level of caution as with any other
potentially infectious reagent). Lentiviral particles are 3) 100 mM sodium orthovanadate (sc-3540) in frozen
replication-incompetent and are designed to self- aliquots (add at 10 µl/ml RIPA)

Santa Cruz Biotechnology, Inc. 1.800.457.3801 831.457.3800 fax 831.457.3801 Europe +00800 4573 8000 49 6221 4503 0 www.scbt.com
䡲 Subbing solution: 0.3% (w/v) gelatin, 0.05% chromium
potassium sulfate in distilled H2O.
䡲 Tris buffered saline (1x TBS): 10 mM Tris-HCl, pH 7.4;
150 mM NaCl (Tris: sc-3715). Available pre-made in
liquid (sc-24951) form.

Santa Cruz Biotechnology, Inc. 1.800.457.3801 831.457.3800 fax 831.457.3801 Europe +00800 4573 8000 49 6221 4503 0 www.scbt.com

Источник: https://www.scribd.com/document/439508016/Santa-Cruz-Biotechnology-Protocols

Sirna transfection protocol lipofectamine 3000

sirna transfection protocol lipofectamine 3000 Enhanced protein expression using Lipofectamine ® 3000 reagent. Feb 01, 2021 · Lipofectamine 3000 (Thermo Fisher Scientific, USA) was used as the siRNA transfection reagent. Simply substitute siRNA for DNA. 5 μL Reverse Transfection of RNAi Apr 21, 2021 · In a study which compared different non-viral methods for transfecting human iPSC-derived cardiomyocytes (hiPSC-CMSs), Lipofectamine STEM was shown to produce superior transfection efficiency (up to 32%) as compared to other reagents (Lipofectamine 3000, Lipofectamine 2000 and the non-liposomal PEI-based reagents Transporter ™ 5 and PEI25 Apr 20, 2018 · Two days after transfection, the GFP in living cells was monitored using the same lower power setting with 4× objective lens in an Olympus confocal microscope. 5 μL 7. The kit includes: basal medium, FBS, Lipofectamine 3000 reagent, Opti-MEM medium, and TrypLE reagent. 75 µl 0. siRNA Transfection - Transfection Protocol. Follow up the transfection reagent protocol. In addition, It is able to do 3000 x 24-well siRNA trilencers were purchased from OriGene to target TFAP2A (SR304787), PKP2 (SR303544), DSP (SR301285), and JUP (SR302502). Non-targeting siRNA (siNT) was used as a negative control. Protocol Outline. 'Bright Yellow-2' (BY2) protoplasts. Gibco™ DMEM with GlutaMAX™ Supplement 10566016 10% Gibco™ FBS A3160401 Proper culture techniques and procedures are an essential part of ensuring successful transfection. C. Immunofluorescence assay siRNA trilencers were purchased from OriGene to target TFAP2A (SR304787), PKP2 (SR303544), DSP (SR301285), and JUP (SR302502). May 11, 2016 · Lipofectamine reagents are widely accepted as “gold-standard” for the safe delivery of exogenous DNA or RNA into cells. Subculturing, also Lipofectamine® 3000 is a versatile reagent that can also be used to deliver siRNA using the same transfection protocol. The viability and death of transfected cells were 43% and 58% after transfection, respectively. B. com siRNA TRANSFECTION PROTOCOL The method or application for co-transfection is important to help you determine the order of delivery and the right reagent for transfection: 1. 24 h post-transfection, cells were Lipofectamine® 3000 试剂/孔 100 ng 0. For siRNA transfection, Lipofectamine 2000 (Invitrogen) was used according to the manufacturer’s protocol. Versatile —one reagent for DNA, RNA, and co-transfection. To study the effect of siRNA knockdown of the gene that is expressed by the co-transfected plasmid, transfection can be performed at same time by using one reagent, such as Lipofectamine® 3000 or Transfection Amounts Transfection of siRNA To transfect cells with siRNA, follow the protocol as described for DNA but do not add P3000 ™ Reagent when diluting the siRNA (step 3). Lipofectamine RNAiMAX, a new transfection reagent, has been confirmed high efficiency in delivering small interfering RNA (siRNA) into mesenchymal stem cells and neural stem cells. how many ug per ul ? jetOPTIMUS ® is a ready-to-use transfection reagent provided with its own complexation buffer (jetOPTIMUS ® Buffer). Its price is only 1/3 that of L2K, and 1/3. 1. Briefly, mesenteric vessel segments were placed in a petri dish with 1 mL of complete medium containing 10 % fetal bovine serum (Biological Industries, Israel). To study the effect of siRNA knockdown of the gene that is expressed by the co-transfected plasmid, transfection can be performed at same time by using one reagent, such as Lipofectamine® 3000 or Feb 01, 2021 · Lipofectamine 3000 (Thermo Fisher Scientific, USA) was used as the siRNA transfection reagent. For this transfection, 6. 3801 831. Transfection Amounts 96-well 24-well 6-well Final siRNA used per well 1 pmol 5 pmol 25 pmol Final Lipofectamine® RNAiMAXused per well 0. B: 25 μL Lipofectamine transfection reagent is diluted with 100 μL serum-free medium. g Feb 01, 2021 · Lipofectamine 3000 (Thermo Fisher Scientific, USA) was used as the siRNA transfection reagent. 3801 Europe +0080045738000 49622145030 www. siRNA transfection. Transfection protocols are used in biological laboratories for introduction of foreign plasmid DNA, small therapeutic RNA, or protein molecules into cells of a eukaryotic nature. TRANSFECTION PROTOCOL Lipofectamine 3000 Transfection Reagent MCF10A Breast cancer cells Complete growth medium Component Cat. 5 µL siRNA转染 有限产品质保 重要授权信息 免责声明 Lipofectamine® 3000试剂实验方案 转染siRNA至细胞中时,遵循如上所述的DNA实验方案,但在稀释siRNA时不要 加入P3000™试剂(第3 DNA-In™ has a simple one-tube protocol and the price is comparable to, or lower than, most reagents on the market. Lipofectamine® RNAiMAX Transfection Protocol See page 2 to view a typical RNAiMAX transfection procedure. 15 和0. Broad Spectrum DNA/siRNA delivery - one transfection reagent and protocol for a variety of cells. Jul 22, 2020 · In a study which compared different non-viral methods for transfecting human iPSC-derived cardiomyocytes (hiPSC-CMSs), Lipofectamine STEM was shown to produce superior transfection efficiency (up to 32%) as compared to other reagents (Lipofectamine 3000, Lipofectamine 2000 and the non-liposomal PEI-based reagents Transporter ™ 5 and PEI25 Lipofectamine 2000 CD Transfection Reagent: Same performance as Invitrogen Lipofectamine 2000, certified animal-origin free (“CD” = chemically defined) Lipofectamine LTX & Plus Reagent: High transfection efficiencies and viabilities in common cell lines, particularly Chinese Hamster Ovary (CHO). 75 和7. siRNA trilencers were purchased from OriGene to target TFAP2A (SR304787), PKP2 (SR303544), DSP (SR301285), and JUP (SR302502). Limited Product Warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies’ General Terms and Lipofectamine® 3000 试剂/孔 100 ng 0. Lipofectamine 3000 reagent performs significantly better than leading competitor reagents. 5µl 0. . It showed the highest GFP expression using Lipofectamine 3000 transfection reagent (A) as compared with using Lipofectamine 2000 (B) or FuGENE 6 (C) transfection reagents. No. 5 µg plasmid DNA and volume of one µl of lipofectamine 3000 reagents were identified for 95% transfection efficiency in the KYSE-30 cell line. Transfection Protocol TRANSFECTION PROTOCOL. To demonstrate the performance of DNA-In™, we directly compared transfection efficiencies and toxicity of DNA-In™ against Lipofectamine 2000 and 3000, using several common, well-established cell lines, primary Lipofectamine 3000 is the best choice for transfection of CHO-K1 and HEK293 with pCDH while Turbofect is preferably used in transfecting these cell lines with pEGFP-N1 (Tab. (B) Summary of increases in titer levels using two different cell lines and insert sizes. However, regardless the transfection reagents the overall transfection efficiency was low and in SH-SY5Y cells (<40%) and further experiments are Feb 01, 2021 · Lipofectamine 3000 (Thermo Fisher Scientific, USA) was used as the siRNA transfection reagent. Nov 29, 2021 · Cells were transfected with siRNA targeting Atg5, Atg7 or PIK3CA using Lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer’s protocol. 457. 800. The siRNAs were prepared as 20 μM stock solutions in diethyl pyrocarbonate–treated water. However, regardless the transfection reagents the overall transfection efficiency was low and in SH-SY5Y cells (<40%) and further experiments are Mar 02, 2020 · The lipofectamine 3000 and LTX transfection for virus production was combined with concentrating the resulting lentivirus supernatant by means of an ultrafiltration spin-column as well as gentle pelleting of the recipient cells prior to transduction; yields were compared to results obtained with a commonly applied protocol using PEI (e. A. Despite this, a satisfactory mechanism-based explanation of their Specially designed cationic lipids, such as the Invitrogen Lipofectamine Transfection Reagents, facilitate DNA and siRNA delivery into cells (Chesnoy and Huang, 2000; Hirko et al. A western blot was performed to determine the level of expression using -actin as a control. For this experiment, knockdown of endogenous luciferase was achieved in three engineered luciferase cell lines using Lipofectamine ® 3000, Lipofectamine ® 2000, and siRNA transfection with Lipofectamine 3000 is believed to yield very high transfection efficiency (higher than any other commercial transfection reagent available with very precise reproducibility). 5 μg 6h 0% 24h 0% Transfection percentage was determined by eye after 24 hours and best condition were achieved using K4 Transfection System 1:2 (μg/µl) replacing the media after 6 hours of incubation. Improved cell viability —gentle on your cells, with low toxicity. 5 nM. Detailed protocol as following: siRNA trilencers were purchased from OriGene to target TFAP2A (SR304787), PKP2 (SR303544), DSP (SR301285), and JUP (SR302502). Achieve Superior Transfection Efficiency While Maintaining Low Toxicity. Final concentration of siRNA was 12. 25 pmol of siRNA in 50 µL of media without serum was mixed with 50 µL of media without serum containing 1. In this study, we used three transfection reagents (Lipofectamine RNAiMAX, Oligofectamine and Lipofectamine 2000) to deliver siRNA into human embryonic stem (hES Broad Spectrum DNA/siRNA delivery - one transfection reagent and protocol for a variety of cells. 5 µL of Lipofectamine 3000. Lipofectamine® 2000 (Thermo Fisher Scientific) and 25 nM siRNA according to each manufacturer’s protocol. HepG2 cells were transfected with a vector expressing GST-tagged STAT protein. Briefly, 1 day prior to transfection, cells were seeded without antibiotics so as to be 50% confluent at the time of transfection. Lipofectamine Transfection Reagent Oct 01, 2016 · The liposomal-based Lipofectamine2000 (L2K) and Lipofectamine 3000 (L3K), which allegedly works equally well for both siRNA and plasmid DNA, and the polymer-based TransIT-X2 TransIT-TKO, TransIT-siQUEST, X-tremeGENE siRNA, Xfect mESC and Nanofectin siRNA were evaluated. The siRNA oligomer–Lipofectamine 2000 complexes were prepared by Lipofectamine 3000 and RNAiMAX outperformed Lipofectamine 2000 in HEK 293 cells (Figure S1 Metzger Lab Protocol Book 12/1997 LipofectAMINE Transfection of CHO Cells 1. Prepare plasmid DNA-lipid complexes (recommend 2 doses of lipid). 3800 fax831. Nov 30, 2021 · When cells reached a confluence of approximately 50–70%, they were transiently transfected with 500 ng of the RDH14-pDEST53 vector using Lipofectamine 3000. 5mL size provides up to 1500 transfection reactions (in 24-well plates). Santa Cruz Biotechnology, Inc. Cells were incubated for an additional 48 h and were then collected for subsequent experiments. Gibco™ Ham’s F-12 Nutrient Mix 11765054 5% Horse Serum – 1 mM Gibco™ Sodium Pyruvate 11360070 25 mM Gibco™ HEPES 15630106 Proper culture techniques and procedures are an essential Lipofectamine ™ 3000 Reagent Protocol. 26). Aug 03, 2012 · Avoid fluorescent siRNA controls when working at low-siRNA concentration since high-siRNA concentration is required for detection (20–50 nM). Welcome to siRNA transfection resource. Transfection Amounts Transfection of siRNA To transfect cells with siRNA, follow the protocol as described for DNA but do not add P3000 ™ Reagent when diluting the siRNA (step 3). Transfection Amounts Transfection of siRNA. Mar 10, 2014 · The method or application for co-transfection is important to help you determine the order of delivery and the right reagent for transfection: 1. (A) Titers obtained using Lipofectamine 3000 reagent compared to commonly used transfection reagents in 293FT cells using a 3 kb insert gene. Lipofectamine® 3000 reagent is designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cell types. Immunofluorescence assay Feb 01, 2021 · Lipofectamine 3000 (Thermo Fisher Scientific, USA) was used as the siRNA transfection reagent. siRNA Transfection siRNA Transfection – Protocols, techniques, methods, in vivo transfection. , 2003; Liu et al. Transfection efficiency is close to that of Avalanche®-Omni Transfection Reagent, but with much lower prices. Cells transfected with Lipofectamine 3000 served as control for comparison. 2 µL 0. Plate cells so they will be 70–90% confluent at the time of transfection. For this experiment, knockdown of endogenous luciferase was achieved in three engineered luciferase cell lines using Lipofectamine ® 3000, Lipofectamine ® 2000, and ** Optimum amount needed is determined from the protocol for Lipofectamine 3000 Transfection Reagent. 2, Ref. For your convenience, the essential components of this protocol are now available in the Gibco™ Breast Cancer Starter Kit. 11668027)Thermo Fisher lipofectamine 2000 protocol Lipofectamine 2000 Transfection Reagent is a versatile transfection reagent that has been shown to effectively transfect the Nov 29, 2021 · Cells were transfected with siRNA targeting Atg5, Atg7 or PIK3CA using Lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer’s protocol. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. A green fluorescent protein (GFP) reporter was fused in-frame with the C-terminus of the Cas9 protein and the fusion protein was successfully delivered into non-transgenic tobacco cv. 3 µL 500 ng 1 µL 0. Mix solution A and solution B, let rest about 30 min at room temperature. 1 day ago · Two lipofection reagents, Lipofectamine 3000 and RNAiMAX, were adapted for successful delivery into plant cells of Cas9/gRNA RNP. In biomedical applications, this process of RNA interference has encouraged researchers to study ways in which this process can be utilized to shut down or effectively incapacitate a defective or non-wanted gene’s ability to replicate. Add DNA-lipid complexes to cells. Lipofectamine® 3000 is a versatile reagent that can also be used to deliver siRNA using the same transfection protocol. 3 μL 1. To transfect cells with siRNA, follow the protocol as described for DNA but TRANSFECTION PROTOCOL Lipofectamine 3000 Transfection Reagent HepG2 Liver cancer cells Complete growth medium Component Cat. Limited Product Warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies’ General Terms and When the cells reach confluence, Lipofectamine 3000 transfection reagent was used to transfect A549 cells with fluorescent plasmids containing IL-32 siRNA. CGU. The 1. , 2003). Figure 1. METAFECTENE PRO and Lipofectamine 3000 reveals that K4 Transfection System is the most effective transfection reagent for SH-SY5Y cells followed by METAFECTENE PRO and Lipofectamine 3000. Twenty-four hours after siRNA transfection, cells were treated with or without dihydroartemisinin for another 24 h. Lipofectamine® 3000 Transfection Reagent Lipofectamine ® 3000 reagent leverages our most advanced siRNA trilencers were purchased from OriGene to target TFAP2A (SR304787), PKP2 (SR303544), DSP (SR301285), and JUP (SR302502). Jan 20, 2020 · The Optimized concentration of 1. 75 和1. scbt. 5 µL 2500 ng 5 µL 3. 1, Fig. In addition, It is able to do 3000 x 24-well Lipofectamine 3000 0. Forty-eight hours after transfection, the IL-32 shRNA transfection was evaluated by observing the intensity of green fluorescent protein with an inverted fluorescence microscope. The basic structure of cationic lipids consists of a positively charged head group and one or two hydrocarbon chains. When around 80% unilaminar cells formed at the bottom of culture flask, wash cells two times with serum-free medium and add 1 mL serum-free medium each well . CRC cells were transfected with siRNA against c-Myc (Santa Cruz Biotechnology) with Lipofectamine 3000 (Lipofectamine™ RNAiMAX) transfection reagents according to the manufacturer’s instruction (Invitrogen). 4 that of Lipofectamine® 3000 (L3K). 5 µL siRNA转染 有限产品质保 重要授权信息 免责声明 Lipofectamine® 3000试剂实验方案 转染siRNA至细胞中时,遵循如上所述的DNA实验方案,但在稀释siRNA时不要 加入P3000™试剂(第3 Lipofectamine 3000 is the best choice for transfection of CHO-K1 and HEK293 with pCDH while Turbofect is preferably used in transfecting these cell lines with pEGFP-N1 (Tab. 5 μg 6h 0% 24h 0% P3000 Reagent 1µl Lipofectamine 3000 1. sirna transfection protocol lipofectamine 3000

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