Protein Crosslinking - Tryin' to Crosslink 2 proteins using -SH group (May/29/2007 )
I am trying to crosslink 2 bacterial proteins using their exposed -SH groups (I had incorporated the cysteine mutations). Now these proteins interact with each other (the crystal structure of complex not known) and the cysteine mutations are very near the hypothesized binding sites. I want to crosslink the two cysteine mutant proteins and send for Mass-spec analysis. But, I didn't find any crosslinking reagent that would minimize the probability of these two different protein getting crosslinked to themselves rather than binding to their counterparts.
Can anyone suggest me about this issue?
Unpair SH groups are unstable, and will interact with other SH groups nonspecifically. I guess keep it in the free state as long as you can with reducing agents, mix and allow them to interact with each other, then cross-link them right after you pass them through a spin column to remove low MW reducing agents. Flash N2 to keep O2 away, use EDTA to neutralize heavy metal ions, and do it as quickly as you can. Bis maleimide cross-linking agent may work in principle. Pierce has it, I believe.
Thanks a lot! But, during the cross linking reaction is there a way to minimize the two different proteins crosslink within themselves and allow only to pair with their counterparts? I know that I can use HPLC to separate the self crosslinked proteins.....just want to minimize it from happening.
I did lots of crosslinking a few years ago. Try cross-linkers of different spacer arm lengths. I've used things like APA-Br (9A linker) and APDP (21 A linker). They all have one thiol-specific group, and one non-specific, photoactivatible group. There are heaps more out there. You'll get very different results according to which one you use, because of the linker length and the orientation of the groups. Hope that helps!
I have done quite a bit of crosslinking in the past. There is no way to favor intermolecular crosslinks vs intramolecular. Chemically, these species are the same and the intramolecular is often favored because sulfhydryls there are locked in proximity to one another. Youjust have to expect both types of crosslinks and plan to deal with them.