Hi,
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?
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2 replies to this topic
#2
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Posted 13 November 2009 - 12:58 PM
the HA tag protein is so small ~ 1-2 kd so might need to use a suitable gel and markers
#3
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Hmmm, I think it's working
Posted 15 November 2009 - 04:01 PM
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.
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.
Edited by bob1, 15 November 2009 - 04:01 PM.
