I am working as a PhD student in a medical biochemistry department.
I am planning to do a crosslinking experiment but I have some chemical problems.
I want to crosslink a protein with an interacting peptide to investigate the site of interaction. The protein is around 200kDa big and has 4 subunits. The peptide is 28aa long and has 3,4kDa. There is a sulfhydryl-group at the N-terminal-end of the peptide and several amine-groups in the middle.
I want to use the SH-group at the end because I do not want to disturb the protein-peptide interaction too much. The idea is to couple a heterobifuncional crosslinker to the SH-group of the peptide, then allow the peptide to interact with the protein and then crosslink the protein to the peptide via amine-groups of the protein.
So the crosslinker has to react first with the SH-group and later on with an amine-group. Is this possible??? Do the reaction conditions and properties allow it?? I already try to find a crosslinker that acts like this, but I just found the other way around (first amine, second sulfhydryl).
So my questions are:
Do the reaction conditions allow a crosslinking first with a SH-group and later on with an amine-group? What crosslinker is possible?
It would be very great if someone could help me.