2 replies to this topic

[cocco]

I have overexpressed a protein (pI = 5.34; charge at ph7 = -17.7) with a chitin fusion on its N-terminus.  Here's the problem - after running an SDS-PAGE, the size of the overexpressed protein (prior to cleaving the chitin tag, from induced bacterial culture) is correct, approx. 35kDa.  However, post-cleavage and purification, I obtained a significant band, size approx. 60kDa.  I haven't carried out a Western blot with the appropriate antibody yet (will be doing it over the course of this week), but an enzymatic assay with the end product have shown a decrease in the substrate level (in other words, it seems to behave like what the target protein should).  Would be eternally grateful to anyone who can help / advise / drop an idea on why the SDS-PAGE results is behaving in such a boggling manner!  

Cocco x

[HomeBrew]

If it's a very hydrophobic protein, maybe it's forming a dimer?

[Prep!]

i second tat... try doing a reducing gel... if its not disulphide linked then try treating it with urea or Gu HCl and then run a nom-reducing gel!!
Best luck!