Protein extraction from perfusion fixed brain tissue - (Dec/14/2010 )

Hi,

I`m trying to extract proteins from perfusion fixed brain tissue to do Western blot.
I`ve tried to use a standard RIPA lysis buffer with protease inhibitors. So I homogenized a piece of
tissue in the buffer and incubated it for 30min on ice. Next I centrifuged it and took the supernatant.

I`ve done a BCA-test and got high ratings (so high protein concentrations)
Next I performed a standard beta-actin western blot but was disappointed by the result.
Just a really thin, unclear band for Beta-actin and lot of `rubbish`at the bottem of the lane.

Does this mean that my proteins are broken down and that I can not measure them anymore?
Does anyone have a different buffer/approach to get these proteins from perfusion-fixed tissue?
THANK YOU!

-Blackeyed-

Pefusion fixed with what? Formaldehyde cross-links the protein so that it is very difficult to break down or solubilise.

-bob1-

4% Paraformaldehyde perfusion and then perserved on sucrose

-Blackeyed-

Is it then an idea to add more SDS to the lysis buffer and maybe boil the samples?

-Blackeyed-

No, I think you will need a stronger system than just SDS, probably some urea and pH alteration will be necessary. Have a browse on the web, there should be a few protocols around.

-bob1-

What does the urea do then?
Do you have an example of such a protocol?

-Blackeyed-