Does anybody have experience with ACC (Acetyl Coenzyme A Carboxylase) and pACC detection by western blotting? It is often used to confirm the status of AMPK activity. The antibodies we use are from Cell Signaling Tech. and they are cited in several publications. However, our anti-ACC does not work at all, while anti-pACC recognizes an unspecific sharp band at 100 kDa and a signal, which is running around the expected MW 265 kDa, not as a defined band, but as a “diffused zone”. It should be pACC, because its intensity mirrors that of pAMPK. I am not sure, if it is a problem related to our SDS-PAGE 6% gels or blotting or lysate preparation (RIPA buffer + protease and phosphatase inhibitors, then treated 5 min at 95 °C in highly reducing conditions (beta-mercaptoethanol, 5% in lysates). We are trying to detect ACC in HepG2 cells and rat liver lysates. This is really frustrating, because according to several publications pACC and ACC should be sharp bands. Please help us to identify the critical problem and get nice pACC/ACC blots.
Hi Clara, thanks a lot for your suggestion. It is It is suprising that you load your samples on 10% SDS-PAGE, because for such a high molecular weight I would use low percentage or gradient polyacrylamide gels. You probably need to run them quite some time to make sure that ACC enters the gel and runs down few mm. How long do you run a standard 10% mini gel? Where can I find the receipe for your lysis buffer? Maybe changing lysis conditions will help ...
Dear Elzbieta
The SDS could interfere with blot. Generally, the presence of the strong, anionic detergent SDS on the membrane or in buffers can result in non specific band development caused by antibodies binding to the charged SDS molecules associated with the proteins. I suggest you to ensure that the blot is washed extensively after transfer and no SDS is used during Western blot development.
Marcellin.
Hi Stephanie, we figured out that ACC migrates only partially into the gel and most of it remains in the stacking, or more precisely at the boundary of stacking and running gels. The solution is to use a gradient gel, such as 4-12% acrylamide. You can buy them from Biorad and run your samples in standard Laemli buffer. In alternative, Invitrogen NuPage gels work even better. I am sure however, that the chemistry of our home-made SDS-6% acrylamide combined gels has some problems ..., so we solved them for now by buying pre-made gradient gels.
Thanks to Clara and Marcellin who tried to help to find a solution.
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