Hello, I am trying to dialyze a protein I'm working with in a buffer containing 100 mM L-cysteine.
From what I've read, L-cysteine should be dissolved in a low pH solvent, in an airtight environment since it is hygroscopic. After making a high concentration solution of cysteine (~500 mM), would transferring a large volume of it into a buffer solution, along with adding a dialysis cassette compromise the L-cysteine? Would I have to perform all of this in an anaerobic hood or is there another way?
Thank you.
Why are you using such a high concentration?
Be aware that you will likely get cysteinylation of your thiols!!
I agree with above: such a high concentration is unusual and could lead to strange disulfide reactions.
Your comment about perhaps using an anaerobic hood suggests you are worried about oxygen. I work with oxygen-sensitive solutions. My recommendation for large volumes is to sparge (bubble) with nitrogen to purge dissolved oxygen from the water that you will use to make the buffer. And then continue bubbling nitrogen into the dialysis bath. If you cover (not airtight) the entire bath, the positive pressure from flowing nitrogen will keep out atmospheric oxygen, and it takes only a fairly low N2 flow to maintain that situation.
If you want to make sure that you have full reduction without derivatization of the molecule, use DTT. You can use much lower concentrations than the 100mM with cysteine; DDT's is a more potent reducing agent.
As others have said, 100 mM Cys is very high. If you want to use it as a reducing agent for proteins, 0.1 - 1 mM is sufficient (and, as Achim Recktenwald pointed out, DTT at the same concentration would be better, see doi:10.1021/bi00892a002); if you want it as buffer substance, 10-20 mM should usually do. The pKa value of the carboxy-group of Cys is 1.96, of the amino group 8.18. You therefore could use Cys to buffer between pH 1-3 or 7-9. The pI of Cys is hence 5.07, at this pH it is least soluble. The -SH group has a pKa of 10.28, suitable for a pH-range of 9-11.
Cys can be oxydised (R-SH + HS-R R-S-S-R + 2 [H]) for example by air oxygen. This, rather than hygroscopy, is the likely reason for excluding air.
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