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Protein Isolation from mouse brain: problems

protein isolation brain mouse pellet Help

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#1 tribio

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Posted 18 April 2012 - 08:42 AM

Please help.
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.

#2 bob1

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Posted 18 April 2012 - 01:35 PM

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.

#3 Inmost sun

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Posted 22 April 2012 - 02:54 AM

I would spin at 100.000 xg





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