I have acquired formalin fixed tissue (adult rat hippocampus) for which I am trying to extract proteins and seperate on a western with subsequent antibody application etc. I have used a couple of different methods to extract the proteins:
1. Sonication, Lysis buffer, 100c incubation 20min, 60C incub 2 hr...western
2. Sonication, SDS 2.5% detergent buffer, 100C incub 20, 60C incub 2 hr...western
These are derivations of Ikena et al 1998 protocol.
I am achieving a positive result on Coomassie protein assay, low but the protein is there; however, when I run the gel NO protein is trasferred to the membrane (ID with Ponceaus). Does anyone have any suggestions for westerns with formalin fixed tissue?