trizol extraction for phospho proteins
Posted 23 January 2005 - 06:15 PM
Does anyone know if a phenol based extraction would be too harsh when looking for phosphorylated proteins?
Any help would be much appreciated.
Posted 23 January 2005 - 06:21 PM
Posted 27 January 2005 - 10:45 AM
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!
Posted 30 January 2005 - 05:49 PM
Thanks again for your reply
Posted 31 January 2005 - 01:55 AM
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
Edited by fred_33, 31 January 2005 - 01:58 AM.