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Protein extraction from perfusion fixed brain tissue


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#1 Blackeyed

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Posted 14 December 2010 - 05:42 AM

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!

#2 bob1

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Posted 14 December 2010 - 04:59 PM

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

#3 Blackeyed

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Posted 15 December 2010 - 02:04 AM

4% Paraformaldehyde perfusion and then perserved on sucrose

#4 Blackeyed

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Posted 15 December 2010 - 06:10 AM

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

#5 bob1

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Posted 15 December 2010 - 03:58 PM

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.

#6 Blackeyed

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Posted 16 December 2010 - 04:25 AM

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




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