Cleavage of GST-tag with Factor Xa - (Mar/13/2013 )
My GST-tagged protein has a Factor Xa site between the tag and the protein. In order to optimise conditions I have tried incubating 0.5U to 10U of Factor Xa with 5uM of my protein for time periods of 2-16 hrs. But when I run the samples on SDS-PAGE, I do not see anything. The gel looks like there was absolutely no protein loaded. I am unsure as to what is going on. Please help.
Does your protein show up on SDS-PAGE before cleaving with FXa? If it does and then disappears after adding FXa, it's possible FXa could be cleaving your protein at additional sites, check the sequence to see if there might be other possible cleavage sites.
Also, GST is a pretty big tag (~26kDa), so will change the size of your protein significantly, so make sure your SDS-PAGE conditions are optimal for the cleaved size protein and it's not running off the gel. You should see both the cleaved protein and the GST tag though.