trizol extraction for phospho proteins - (Jan/23/2005 )
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.
sorry - i just realised I should have put this on the protein page!
Hi Nay, check this link
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!
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
I use a simple method for extracting proteins from cells.
Here is te protocol:
use 300µl of lysis buffer for 5 millions of cells
incubate cells + lysis buffer 10' on ice
Centrifuge 14000 rpm 30' 4°C
discard the pellet
the extract is ready for quantification and more analysis
sample buffer :
NaCl 150mM (3ml NaCl 1M)
EDTA pH8 2mM (160µl 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 :
-5µl DTT 1M
-1µl PMSF 100mM