Say, after protein quantification, we mix certain amount of samples, loading dye and reducing reagent, what should be used for bringing up the final volume, deionized H2O or lysis buffer? Is Lysis buffer better to keep the detergent condition stable for protein of interest?
Thanks ahead for providing answers.
You should use the lysis buffer used for protein extraction.
@ Milica, Right? That is what I thought. Somehow, it is H2O in the protocol from manufacture's instruction, which I followed. Now when I tried to demo to my students, all of sudden I was thinking whether I should use lysis buffer rather than H2O. But just not too sure about it. Will that introduce too much salts in the sample by using lysis buffer?
I wouldn't bother diluting. IF you've quantified your protein for Western Blotting, just calculate for the individual wells. if you dilute, it makes good sense to recheck concentration, and if they didn't dilute as expected, your PI will not be happy with you and your students will be confused...that and in my experience smaller volumes loaded to wells give tighter bands.
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