Bacterial expression antibody recognition problem - antibodies won't recognize protein (Sep/29/2008 )

Hi!

I'm trying to express a protein using BL21-AI cells and the pDEST17 vector which has an N-terminal his tag. I've been able to express a protein of the correct molecular weight which is recognized with an antibody to the His tag. However when I try to check by western with an antibody to my protein (I know that the antibody works, however it was raised against the n-term part of the protein) I don't see anything on the western from my expression. I've checked the sequence, everything seem perfect- in frame, correct sequence, etc. I've tried blotting with other antibodies to the protein and nothing seems to recognize it. Can anyone suggest any trouble shooting before switching to another vector with a c-terminal tag?

thanks!

If the protein's tertiary structure is so screwed up that many of its epitopes are gone, it's likely not going to retain enzymatic activity anyway. Were you purifying the enzyme for in vitro enzymatic assays?

It's also possible that the SDS-PAGE is denaturing the protein to such a degree that the antibody can no longer recognize it. Have you tried a dot blot?