SDS and urea lysis, protein degradation and freeze-thaw cycles - (Aug/30/2013 )
I have three possibly naïve questions:
1a) If we utilize 1% SDS to lyse cells, would we need to add protease/phosphatase inhibitors? Given that proteases/phosphatases need to be correctly folded to function and SDS effectively denatures everything, would adding inhibitors be overkill?
(I am aware that proteinase K activity is enhanced by SDS, but I'm only interested in endogenous proteases/phosphatases)
1b) Would this also apply to high molar urea buffers? (e.g. 8M urea).
2) Imagine we have a cell lysate (in 1% SDS): I've seen people concerned about how many freeze-thaw cycles lysates are subjected to, given that it 'damages' proteins. How can freeze-thaw cycles damage completely denatured proteins? Would ice crystals somehow brake the peptide bond? Same concern with phospho-proteins...I've heard people say that freeze-thaw cycles will 'chop off' phosphates. Are these concerns warranted or just 'lab culture' we all inherit without any rationale behind?
Thanks for your time!
a) Yes, they are quite stable proteins and can still be active in 2% SDS.
b)Don't know, but suspect not.
2)They are warranted - you can test this for yourself, take a lysate and freeze/thaw it 10 x and see how it looks on a gel or blot compared to a single freeze/thaw.
Hey bob1, thanks for your reply.
In regards to your (a) answer, I've been looking around Pubmed and google scholar for publications that reference proteases/phosphatases that resist SDS but can't find any. Could you send me a link?
I don't have any references, but a bit of experimentation will give you some empirical evidence.
references of that type may be difficult to find. you would have to look up articles on the characterization of specific proteases to maybe find such data.
when we were characterizing proteases, we tested activity under a number of conditions, including in the presence of various detergents and freeze-thaw cycles. however, these are not specifically listed in the key words so they won't show up in the search you are requesting.