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trizol extraction for phospho proteins


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4 replies to this topic

#1 Nay

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Posted 23 January 2005 - 06:15 PM

I am trying to look at a phosphorylated protein via western blotting, and have extracted protein from mouse tissues using Trizol (a phenol based solution). However I have been having trouble visualising any bands...

Does anyone know if a phenol based extraction would be too harsh when looking for phosphorylated proteins?

Any help would be much appreciated.

Thanks

#2 Nay

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Posted 23 January 2005 - 06:21 PM

sorry - i just realised I should have put this on the protein page!

#3 badcell

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Posted 27 January 2005 - 10:45 AM

Hi Nay, check this link
http://www.signaling...?pid=PP00000155
they use Trizol for extracting 32P-labelled proteins, so it seems it works. But in my hands is a nightmare to get any protein out of the trizol! :)
Science is a wonderful thing if one does not have to earn one's living at it
(A.Einstein)

#4 Nay

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Posted 30 January 2005 - 05:49 PM

Thanks for that - I haven't had any problems using trizol to extract protein - in fact, it seems to be the only method my antibody likes! But for some reason my phospho-Ab don't like it too much!

Thanks again for your reply

#5 fred_33

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Posted 31 January 2005 - 01:55 AM

I use a simple method for extracting proteins from cells.

Here is te protocol:

use 300l of lysis buffer for 5 millions of cells
incubate cells + lysis buffer 10' on ice
Centrifuge 14000 rpm 30' 4C
discard the pellet
the extract is ready for quantification and more analysis

sample buffer :

NaCl 150mM (3ml NaCl 1M)
EDTA pH8 2mM (160l EDTA 250mM)
NP40 1% (2ml NP40 10%)
Tris HCl 50 mm pH 7.5
ddH2O qsp 20ml

just before use add for 1ml of buffer :
-5l DTT 1M
-1l PMSF 100mM

Edited by fred_33, 31 January 2005 - 01:58 AM.





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