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:
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?
This will be difficult because formalin is a cross-linking fixative, the
proteins of the cell will be chemically turned (I exagerate of course)
into one big molecule. You may have much better luck by embeding and
cutting the tissue and then using immuno-histochemistry. This will also
require much less of the sample.
There have been recepies for digesting formalin-fixed tissue to make it
more reactive in histochemistry: Microwaving in citrate buffer, gentle
protease treatment and the like. But to expect proteins with defined
molecular weight, required for western, to come out of this would be...