Protein Isolation from mouse brain: problems - (Apr/18/2012 )
I am having problems with protein isolation from brains of mice. After centrifugation my pellet is slimy, stringy and noncompact. What do you suggest I may be doing wrong? What will help with this problem?
Protocol: Remove brain and freeze half at -80 degrees Celcius. Remove and homogenize brain in protein lysis buffer (50mM Tris-HCl, 1% Igepal, 1mM EDTA, 1mM Na3VO4, 1mM NaF, 10% glycerol, 1mM DTT, 4.5mM NaPyrophosphate, 10mM beta-glycerophosphate, 1 Roche complete Mini tablet with EDTA). Incubate on ice for 30 min. Spin at 14000 RPM for 30 min at 4 degree celcius. Remove supernatant containing protein.
I am having a very hard time with the last step as my "pellet" is interfering.
Thanks for your help.
It sounds like you have problems with the DNA. You could try sonicating or passing the solution (up to 50x) through a fine hypodermic needle to break up the DNA.
I would spin at 100.000 xg