Checking recombinant protein size - (Jul/09/2008 )
Hi,
I am using a commercially available recombinant protein which is mutated to prevent cleavage. However, my results lead me to suspect that it is, in fact, being cleaved. What would be the best way to check which proteins are present? I'm not sure whether I can run a regular protein gel and be able to detect proteins present, since I cannot incorporate S-met etc as when translating proteins in vivo/vitro. Any help would be gratefully received!
Western blot it. It may already have a common epitope tag (i.e. 6His), so it could be easy to find good antibodies.
I am using a commercially available recombinant protein which is mutated to prevent cleavage. However, my results lead me to suspect that it is, in fact, being cleaved. What would be the best way to check which proteins are present? I'm not sure whether I can run a regular protein gel and be able to detect proteins present, since I cannot incorporate S-met etc as when translating proteins in vivo/vitro. Any help would be gratefully received!
If the quantity of the protein is high enough, then you should be able to run an SDS PAGE gel and stain it with coomassie or silver stain to detect the proteins that are present.
I suspect what you're looking for is not just a way to track the presence of the protein, but rather an explanation as to why it is being cleaved when it's supposed to be resistant to cleavage.
Do you suspect that the cleavage resistance mutation is not present in your protein prep? You will likely not be able to differentiate between mutant and non-mutant forms of the protein by western blotting or Coomassie staining, unless the mutation is significant.
Can you tell us more about the protein you're using? What type of proteolytic cleavage is it resistant to? What is the nature of the mutation that confers this resistance?