Flag tag not working, although theoretically it's there - (Jul/03/2008 )
The protein I work on has a flag tag at the N-terminus. I did a C-terminal deletion mutant of the protein. I checked the sequence and everything is correct: the N-terminus is identical to the original protein, the tag is there etc. The problem is that the tag is not recognized by a Flag antibody (checked 1 antibody and flag coIP beads). I really need it to work, I want to have both proteins (wt and deletion) tagged the same way for IP experiments. Any ideas why it doesn't work?
I would really appreciate your help!!!
thanks
j
-josephine-
QUOTE (josephine @ Jul 3 2008, 02:05 AM)
The protein I work on has a flag tag at the N-terminus. I did a C-terminal deletion mutant of the protein. I checked the sequence and everything is correct: the N-terminus is identical to the original protein, the tag is there etc. The problem is that the tag is not recognized by a Flag antibody (checked 1 antibody and flag coIP beads). I really need it to work, I want to have both proteins (wt and deletion) tagged the same way for IP experiments. Any ideas why it doesn't work?
I would really appreciate your help!!!
thanks
j
I would really appreciate your help!!!
thanks
j
Perhaps the protein is folding differently and the flag tag is now getting buried so that it is not recognized by the antibody. Although, i would also make sure that it's being expressed. Do some RT-PCR to confirm. Perhaps by deleting some c-terminal sequences you also deleted regulatory sequences and its not being processed properly (either transcriptionally or translationally).
-smu2-