I have a protein with a 10xHis-MBP tag on the N-terminus. I'm fairly certain I am expressing my protein, but I'm not getting a signal on my western blot. Is there a possibility that the 10xHis tag is preventing the anti-MBP antibody from binding to MBP?
Dear Ansley
With protein all is possibile but i do think that the his is the problem.
Also because, it is difficult but theoretically possible that the His tag mask the anti-MBP epitope when protein is folded (eg in ELISA) but i do not think that is it may happen in WB, in which the protein is unfolded.
Is your anti-MBP antibody monoclonal or polyclonal? And if it is monoclonal it is certified for use in WB? Because you can also have the possibility that if you are using an antibody that recognize a conformational MBP epitope, it will not work in WB.
good luck
Manuele
Can you observe the targeted protein band as expected at PAGE analysis and are you performing the WB with the protein sup. (before the purification) or with the purified sample using the tags?
Hi Ansley Scott
To check if you do have protein expression, you could alternatively stain for the His tag signal using an antibody. If you have no expression, then your protein is most likely not being expressed. If you do have expression, then you will need to confirm that that is from your protein.
How are you producing your protein? If you run a control without induction and your sample with induction, you should see the additional band appear that tells you your protein is expressed.
Best of luck!
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