Western Blotting Protocol

In this video we take you through all the steps in our Western Blot (WB) Protocol so you can replicate our procedure and get reproducible and reliable results.
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Contents:
0:00 Introduction
1:06 Solutions and Reagents
2:11 Sample Preparation and Protein Blotting
4:33 Membrane Blocking and Antibody Incubations
6:07 Protein Detection
6:45 Importance of Following the Recommended Protocol
8:37 Conclusion
Western blotting is a widely used immunoassay used by research scientists to monitor expression of a specific protein in a complex cell or tissue extract. It utilizes antibodies that recognize a specific protein of interest or a post-translational modification like phosphorylation, acetylation, methylation, and ubiquitination. At Cell Signaling Technology (CST), we perform thousands of Western blots daily using a protocol that has been optimized for over a decade to develop and validate antibodies with exceptional specificity, sensitivity, and reproducibility.
We first list all the the solutions and reagents you’ll need. Then, we give detailed descriptions on how to prepare the sample, perform the protein blot and immunoassay, and detect proteins chemiluminescently. Finally, we describe critical experimental steps in Western blotting and explain how small changes to the protocol, like changing incubation times or dilution buffers, can affect the final outcome of your blot.
To learn more about how to generate publication-ready Western blot data, check out our troubleshooting video ( • Western Blot Troublesh... ). If you’re interested in alternate protocols, including preparing your sample with immunoprecipitation, re-probing your membrane, and detecting your protein using fluorescence detection, visit Protocols at the Cell Signaling Technology website (www.cellsignal.com/protocols).
About CST: Cell Signaling Technology (CST) is a different kind of life sciences company-one founded, owned, and run by active research scientists, with the highest standards of product and service quality, technological innovation, and scientific rigor. We are a company of caring people driven by a devotion to facilitating good science-a company committed to doing the right thing for our Customers, our communities, and our planet. cellsignal.com/about-us
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[@zohasadqat]

this was great for someone like me who hasn't done it yet

[@Polecat-qz5om]

Key word yet 😃

[@JYOtiRaNJanMANgaRaj]

THANK U
FOR A GREAT 😃
ANALYSIS ABOUT WESTERN BLOTTING.
👍👌

[@maretawidiya9672]

like this protocol,.thanks so much

[@arpitkhanna3039]

Thats helpful

[@hkicgh7277]

3:52 After the centrifugation, do you load the samples in the gel without discarding the supernatant layer?

[@cellsignaldotcom]

Correct - this centrifugation step is just to pellet any insoluble material, and pull down any liquid from the side walls of the tube. Load 20 microliters without discarding any supernatant.

[@hanconway8162]

Can you recommend a method for storing pancreatic islets after collection from pancreas for western blot? Can the islets be pelleted and stored at -80? What solution should they be left in until western blot is performed? Thanks

[@cellsignaldotcom]

Thanks for the question, Han.
In general, it depends on both the sample type and the stability of the protein of interest. Ideally, lysates should be freshly prepared, but if this is not possible storage at -20C or -80C may be suitable. If not stored in SDS, the buffer should contain protease and phosphatase inhibitors. Some proteins will yield western blot results with little to no change after repeated freeze-thaw cycles, but other will be affected. You can get in touch with a CST scientist, they may have further information for your sample/protein of interest, please use cellsignal.com/support. You may also want to view our Tech Tips video on lysate buffers - th-cam.com/video/KV51wMtVNak/w-d-xo.html

[@thamizh2850]

On website in the protocol you have mentioned that wash membrane three times for 5 min each after blocking. But here you say only 5 min. So please let me know which one is correct?

[@cellsignaldotcom]

Thanks for the note. After blocking, 3 washes are optional. We typically rinse the excess milk off with TBS-T; one brief wash is sufficient. After antibody incubation, however, three 5-min washes are recommended.

thank you for information

[@p91grwm14]

I have a question regarding the buffer for the western blott. We use normally 20 % methanol in the solution and we have always used nitril gloves in contact with the solution. But currently I have discovered that these gloves are not suitable for methanol. We had never problems but now I'm afraid from that. Is possible that a 20 % methanol solution in water can penetrate the gloves?

[@cellsignaldotcom]

Sorry for the delayed response. It is recommended that you contact the glove supplier and your local EHS or lab safety department for guidance.

[@giahungtran6822]

In the protocol on website, after transfer, wash membrane with TBS 5min. However, in this video, it said that we will wash with TBST 5min. So what is the best choice, TBS or TBST?

[@cellsignaldotcom]

Thanks for the question. Here, either TBS or TBST will work just fine for chemiluminescent WB. Please note that for fluorescent WB, the Tween 20 should be omitted from the optional wash step and the blocking buffer.

[@avanaavan2575]

Do you have offers for students to work western blot in your lab??

[@cellsignaldotcom]

Hi Avana. CST has a summer internship program for undergraduate students and rising high school seniors. Please visit www.cellsignal.com/about-us/social-responsibility/internship-program for information.

[@oliviaxu4492]

is it bad to health while developing x-ray film

[@cellsignaldotcom]

Hi Olivia, x-rays are not part of the Western Blot protocol. If you are developing film manually in a dark room, please use appropriate PPE (safety glasses, gloves, lab coat).