Western Blot Formalin Fixed Tissue

eripearson at rcsi.ie eripearson at rcsi.ie
Wed Jun 23 08:34:17 EST 2004


Help please...

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?

Cheers

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