Expression by ICC but not by Western-why? - (Aug/17/2010 )
I have a problem with one expression construct that I am using: G protein gamma 1 (HA tagged). This works fine in ICC and I can stain cells for HA no problem, but I can't detect anything on a western. The protein is very small (including the tag around 14 kDa) so I have used 15% gels and nitrocellulose and PVDF membranes (0.22 um diameter), different lysis buffers (varying amount of SDS), boiling vs non-boiling of membranes, but nothing works. I tried to blot using the HA antibody and a pan gamma antibody (admittedly from Santa Cruz, so maybe not that great) and still nothing.
Does anybody have any idea what the problem might be and how I can detect my protein? According to my ICC it is expressing at the same level as a G beta subunit that I'm using and I get very high expression with that one.
Can you provide us a detailed western protocol? That might help us see where the problem lies.
Most HA antibodies I have used have a bit of non-specific binding, it could be that some of your signal in the ICC is non-specific.
Having said that, I suspect that part of your problem is the small size of the protein, you may need to do a shorter (and slower) transfer as small proteins can pass through blotting membranes.
Greetings from Santa Cruz.
If you have troubles using one of our antibodies, please contact us in Technical Support here at Santa Cruz. We would be glad to help you troubleshoot your WB results that you are having with your HA tag antibody.
I do agree that it is possible that it is too small and is running off the gel, so I would recommend, just as the previous replier said, to run it slower on the gel.
Feel free to give us a call, we would be happy to help you figure out what is happening on your Western Blot.
Santa Cruz Biotechnology, Inc.
ph: (800)457-3801 ext. 2