I am doing a lot of IHC (immuno histo chem). I have a long list of antibodies which have worked in IHC and I now have to do westerns to demonstrate antibody specificity.
I have begun by testing my rabbit anti-GFAP antibody. I have done a blot in duplicate (5 wells the same on the SDS-PAGE). For a positive and loading control I am using mouse anti-a-tubulin. I have used protein samples (all extracted in RIPA) of rat muscle (which should be GFAP negative), zebrafish brain, goldfish brain (the antibody has worked on both previously in Western Blots) and the test samples (Nothobranchius sp. brain).
On the one half of the blot I only added anti-GFAP. On the other half I added anti-GFAP and anti-a-tubulin. Then I incubated with avadin linked HRP anti-rabbit and anti-mouse antibodies. The secondaries were incubated together. Blocking solution was TBS + tween with 1% milk powder.
I did not get a GFAP positive band on either blot but I did get a a-tublin positive band in all lanes on the blot for which it was probed.
For the IHC I am using PBS.
I am re-running the gel and plan to proceed by trying the following:
1. Using TBS without tween to reduce the specificity. The GFAP protein was of mouse origin and I am working with a fish, Nothobranchius sp.
2. Can I blot in PBS? I have in the past had trouble using the antibodies using TBS as the buffer but others haven't.
Any advice or suggestions would be much appreciated.