ultracentrifugation in membrane protein extraction - (Sep/12/2008 )
Hello,
I want to extract an integral plasma membrane protein from brain for WB. I have found protocols that lyse the cells and then centrifugate about 15,000xg /10 min, while others use ultracentrifugation about 150,000xg / 60 min. I wonder whether it is neccesary to ultracentrifugate. Also in which fraction are the integral membrane proteins?. Thanks a lot.
-zeibe-
Small membranes or vesicles or compartments only come down at very high g's and if your protein of interest is attached to some similar structure, then you need to ultracentrifuge to separate it out. Depending on your experiment, you have to decide if you need to ultracentrifuge or not.
-scolix-
QUOTE (zeibe @ Sep 12 2008, 06:46 AM)
Hello,
I want to extract an integral plasma membrane protein from brain for WB. I have found protocols that lyse the cells and then centrifugate about 15,000xg /10 min, while others use ultracentrifugation about 150,000xg / 60 min. I wonder whether it is neccesary to ultracentrifugate. Also in which fraction are the integral membrane proteins?. Thanks a lot.
I want to extract an integral plasma membrane protein from brain for WB. I have found protocols that lyse the cells and then centrifugate about 15,000xg /10 min, while others use ultracentrifugation about 150,000xg / 60 min. I wonder whether it is neccesary to ultracentrifugate. Also in which fraction are the integral membrane proteins?. Thanks a lot.
I doubt that you can highly enrich plasma membrane by lysis, independent from 15,000 or 150,000 xg centrifugation; cell lysis will also lyse organelles
-The Bearer-