Protein extraction from perfusion fixed brain tissue
Posted 14 December 2010 - 05:42 AM
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?
Posted 14 December 2010 - 04:59 PM
Posted 15 December 2010 - 02:04 AM
Posted 15 December 2010 - 06:10 AM
Posted 15 December 2010 - 03:58 PM
Posted 16 December 2010 - 04:25 AM
Do you have an example of such a protocol?