An electrophilic inhibitor targeting the Cys residue will also react with thiol-containing substances such as glutathione and dithiothreitol at some level. The weak electrophiles that are typically used for this purpose are not very reactive, however, so they may still work even though some thiol is present.

There is 1-10 mM glutathione inside cells, as well as plenty of cysteine, so the covalent inhibitor must be able to work despite that. It would be worthwhile, therefore, to test the effectiveness of your inhibitor with and without the reducing agents present. This will be useful information when you are testing different reactive warheads.

Strictly speaking covalent inhibitors reduce the receptor number. They may bind to the same site as a natural ligand, but this interaction is not competitive, because the binding is not reversible. Competitive interactions tend to be governed by relative KD (dissociation constant) values as ligands bind, accordingly, to available receptors. Reducing agents cause chemical changes, and they are not competitive inhibitors. Their effects may be governed by redox

potentials, and tables exist for some of these.

The DTT and GSH at the concentrations present will ensure that the Cys residues are fully reduced. If you didn't use excess DTT/GSH, they would likely form some mixed disulfides (which you don't want). If your covalent inhibitor is a soft electrophile it will be thiolphilic and likely react readily with reduced DTT, and GSH. I would therefore recommend dialyzing your reduced kinase with a thiol free buffer solution purged with N2 gas (to remove dissolved oxygen, and prevent reoxidation). This solution should be suitable then for reaction with your covalent inhibitor without fear of thiol competition from low molecular weight components (which would occur, if they were present). If you store your reduced now thiol free kinase, do so under nitrogen and keep it cool. You can safely re-reduce it if necessary by adding a little DTT (with just a small molar excess) and then extracting the DTT by partition into diethyl ether, they bubble the solution with nitrogen gas to remove any traces of ether. Avoid contamination with transition metals like iron and copper salts which rapidly promote thiol oxidation. Good luck.