Looking for strong cysteine modification agent? - (May/20/2008 )
I purified a 39-aa small peptide and tried to detect it in PAGE by Coomassie blue. But I found the bands are smear and more concentrated at ~30kD instead of 4kD. This peptide contains 3 pairs of disulfide bonds and very very stable. So before I ran the peptide in gel, I used strong reducing agent, DTT, to reduce it and then use oxidized glutathione to prevent disulfide bond formation inside and between the molecules. But I still see 30kD band instead of 4kD. I suspect that oxidized glutathione is not strong enough to prevent the disulfide bond formation between the peptide molecule, so I saw higher MW band than I expected.
Can you recommend any strong cysteine modification agent to keep my peptide "free" of disulfide bonds and denatured during running in gel? Is there anyone here who has ever faced the similar situation as me? Please advise. Thanks!
maleimide is better. Some ppl use iodoacetic acid as well.
Thanks, genehunter-1. Should I use these agent after the reduction of my peptide by DTT or with DTT? What is the proper concentration of these agents? How long should I let the reaction go? Thanks again.
I am sorry i can't help u. But I would like to ask u for some advice. I have a peptide with a single disulphide bond, and I have been unable to detect it on my membrane. I had not used reducing agent beforehand. Would the presence of the disulphide bond affect the ability of the peptide to enter the gel? How do you make up the DTT and the glutathione? We have L-glutathione (reduced) in the lab; is that what is used?