Dear Colleagues,
I'm working on a recombinant protein (isolated from 3T3 mouse fibroblast supernatants) that behaves a little strange in analytics: Purified by IMAC (it has a His6-Tag) and gel filtration (Sephacryl S200), the 30kD peak is resolving into a monomer and an apparent dimer in SDS-PAGE, regardless if I reduce with BME or TCEP. Upon storage of the sample, the dimer signal gets stronger. When I re-run the material on the S200, also a peak at double size is showing up, suggesting an (apparently very) stable dimer which is not due to SS bond formation and possibly in an equilibrium with the monomer.
Do you have any ideas/suggestions what might be going on, if this is rater an artifact (how to resolve it?) or do/may proteins really exist which are forming dimers/oligomers which are so strongly resistant against reduction and/or boiling with SDS?
Thanks for your comments!
I found an article in which the same observation was made for another protein:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2253441/
Hi, Wo, glad to see you here! I had the same case with some of my constructs, concluding they form so called amyloid bodies; they frequently got stronger when frozen and resist reduction and SDS boiling! BW, Sunny
I have seen it happen with recombinant surface antigen of HBV (HBsAg). You need extended/much more drastic reduction conditions in the presence of a chaotropic agent and heat to get the monomer in SDS-PAGE.
Also, it has been reported that disulfide reoxidation can take place during conventional SDS-PAGE (Multiple bands on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of proteins due to intermolecular disulfide cross-linking, https://www.ncbi.nlm.nih.gov/pubmed/8238894).
Can you not reduce and alkylate with iodoacetamide to find out whether dimer formation is mediated by sulfhydrils in your case?
Thanks for your suggestions, Alejandro! Very useful hints for the analytical part. This at least helps me to demonstrate that I actually have a pure protein, despite is shows 2 bands on SDS PAGE and 2 peaks in SEC.
I haven't mentioned that my actual goal is to use this protein as a probe in mice, so I somehow need to find out what is actually causing this behavior and potentially develop a way to circumvent it.
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