Blocking buffer for phospho-(AKT, GSK3b) WB using Odyssey? - (Feb/25/2013 )
I'm doing phospho-(AKT, GSK3b) WB using Odyssey blocking buffer:PBS 1:1but only see very week signals, and some unspecific bands. I wonder what is the optimal blocking buffer to use. The instruction provided by Licor shows a WB figure comparing Odyssey blocking buffer:PBS 1:1, 5% BSA, etc, but I don't see much difference there. On the other hand I heard that BSA is better than the Odyssey blocking buffer. Does anyone have experience on this? Which BSA concentration to use? (I'm using nitrocellulose membrane)
Thanks in advance.
Basically, the only way to find out is to trial different buffers until you find one that works well. Not all antibodies work well in BSA/PBS and not all antibodies work in the Odyssey blocking buffer either, though most will work in both. You can try adding some tween-20 (0.1%) to the block. Note that you should titrate the primary and secondary antibody dilutions to reduce non-specific bands, don't just rely on the dilution recommended by the company!
If you are using the fluorescent odyssey capabilities, the membrane type you use can have a big effect. It is best to get a fluorescence rated one to ensure that the background is minimized.