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!