Modification of cysteine in my small peptide? - (May/20/2008 )
Hello guys,
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!
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!
Iodoacetamide is often used to alkylate the reduced cysteine residues and prevent disulfide bond formation.
thank you, smoki. I will try that.
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!
Iodoacetamide is often used to alkylate the reduced cysteine residues and prevent disulfide bond formation.