Protocol Online logo
Top : New Forum Archives (2009-): : Protein Expression and Purification

Validating Recombinant Protein - (Oct/17/2014 )

Hi All,

I had a recombinant protein produced by a 3rd part company and it's about 90% pure, according to them. I want to validate it is actually the protein it is supposed to do (and has the right tertiary structure). I tried an ELISA but it didn't show any presenece of the protein. However, I ordered the protein because I don't have a reliable antibody, so the ELISA not working might not mean anything. Anyway, is there any other way I can validate it is actually the protein I wanted? I didn't have a plasmid so they did "sequence optimization" and tested that the protein was present by doing a western with anti-HIS, but that doens't say anything about the protein itself


Sorry for my rambling, is there anyway, without a good antibody, I can validate what I have is indeed the protein of interest? sorry for the ignorance 


Best way would be do a trypsin digest (in-solution or in-gel) and send it for MS/MS analysis. This will unequivocally identify the protein without bias.


Edman sequencing would also tell you if the first dozen or so amino acids are the correct sequence.


Though if a company made it for you, and the company is even mildly reputable, then they've probably got good enough quality control to send you the right thing.


At the very least, run it on a gel to see if it's the right size.