Western Blotting Protocol

[@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. 👍👌

[@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.

[@maretawidiya9672]

like this protocol,.thanks so much

[@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.

[@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 - kzbin.info/www/bejne/gYeYYqqDqbuBl80

[@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.

[@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.

[@arpitkhanna3039]

Thats helpful

[@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.

thank you 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).