Hello all,
I have only done few rounds of western blots using a standard protocol with RIPA. I would like to try to use another buffer
8M Urea
2%CHAPS
2M Thiourea
40mM DTT
1% NP-40
water
I am a bit unsure about the steps of mixing the protein loading buffer (should avoid boiling?), or any specific procedures that are different from using RIPA buffer.
Anyone can share their ways of working? Million thanks!
Tommy: It all depend on what kind of questions you are asking. As your proteins are extracted in the non-ionic NP-40 detergent, Resolution is improved when it is mixed with loading sample containing SDS. It is my experience that a better Western can be achieved if you can couple your primary Ab directly with HRP (see our paper PMID: 25900303).
You must avoid heating urea buffer. For the quantification, you might have some problem due to high amount of urea and chaps.
CHAPS is not suitable for Western blotting as its a non-ionic detergent. But, if you dilute your extract with enough SDS buffer, it could works. you could add some concentrated SDS to replace CHAPS. Often I try to increase SDS up to 4X in final solution !
After transfert, CHAPS is no more a problem and you could do antibodies revelation as usual !
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