protocal for membrane extraction/fractionation - (Jun/01/2007 )
Hi,
I've used the following protocal to extract membrane and it does not works. The pellet is big and viscous, the protein concentration in the membrane part is even higher than the soluable part and the proteins being pulled down using the soluable fraction and the membrane fraction is the same. So I think I simply didn't soluablize the soluable part completely.
The protocal is :
resuspend cells in hypotonoc buffer (only tris and EDTA), votex ,pass
through 22.5 gauge needles 5 times, 100000 g spin 1hr . pellet lysed in TNET (tris
EDTA, NaCl , Trition) will be membrane fraction. Supernant will be
soluable fraction.
Am I doing something wrong? Are there any other methods or protocals ?
How about freeze and thaw cycles, but will it destroy protein protein interactions? How about homogenizing the cells?
Anyone has any suggestions ?
Thank you!
I've used the following protocal to extract membrane and it does not works. The pellet is big and viscous, the protein concentration in the membrane part is even higher than the soluable part and the proteins being pulled down using the soluable fraction and the membrane fraction is the same. So I think I simply didn't soluablize the soluable part completely.
The protocal is :
resuspend cells in hypotonoc buffer (only tris and EDTA), votex ,pass
through 22.5 gauge needles 5 times, 100000 g spin 1hr . pellet lysed in TNET (tris
EDTA, NaCl , Trition) will be membrane fraction. Supernant will be
soluable fraction.
Am I doing something wrong? Are there any other methods or protocals ?
How about freeze and thaw cycles, but will it destroy protein protein interactions? How about homogenizing the cells?
Anyone has any suggestions ?
Thank you!
do differential centrifugation; at least start with 80xg to get rid off cell debris and nuclei;
it is not clear which membranes do you want extract; any membrane or only PM , or ER and Golgi? you may additional centrifugation steps or use sucrose gradient centrifugation to enrich different membrane fractions...
split your questions in distinct posts to increase the chance of replys...
There have been a few posts asking about cells fractionation and membrane isolation. Do a search 
Thank you !