Protein extraction from perfusion fixed brain tissue - (Dec/14/2010 )
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?
Pefusion fixed with what? Formaldehyde cross-links the protein so that it is very difficult to break down or solubilise.
4% Paraformaldehyde perfusion and then perserved on sucrose
Is it then an idea to add more SDS to the lysis buffer and maybe boil the samples?
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.
What does the urea do then?
Do you have an example of such a protocol?