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HA-TAG - HA-Tag- no detection (Oct/21/2009 )

I have HA tagged my protein. My sequencing reaction looks good. Everything should be in frame.
I have added a single HA tag.
The problem is that my protein is expressed (by Western) but I cannot detect it with an HA tag antibody (I use a 3x HA tag for control).
Any ideas?


the HA tag protein is so small ~ 1-2 kd so might need to use a suitable gel and markers


Hmmm, I had a similar problem not long ago... it was the transfection that wasn't working.

My single HA tag works just fine, I am using a Sigma anti-HA rabbit antibody, which shows a strong band of the right size and a much weaker and slightly smaller band in the controls and treatments.

Is there a spacer between the HA and the protein of interest? If not, direct linkage has been known to affect the binding of antibodies to tags.