prepartion of membrane protein - (Jul/02/2005 )
Hi, all,
I am trying to identify the PLC expression in the eggs of marine worms. Since PLC is membrane associated protein, I should use protocol of isolation of membrane fraction. I am not sure I am right or not? Actually, I used the protocol of preparation of the whole cell lysate, but it doesn't work at all. Any suggestions will be helpful. Thanks in advance!
Yin
I'd say try the membrane fraction if the lysate didn't work. Unfortunately i've only prepared membrane fractions of mammalian cells so you'll have to search for protocols specific to your cell type. But if your game to try the mammalian protocol just let me know and I'll send you what I used.
Dr Washo 
Dr Washo
Hi!
In our lab for membrane proteins from mammalian cells, we pass the lysate (before spinning down to remove debris) through a 29 guage syringe 15 - 20 times and then spin down the debris. Seems to work well for us. Try it!
G
[quote=GAYA0002,Jul 4 2005, 06:52 PM]
[quote=dr_washo,Jul 4 2005, 07:41 AM]I'd say try the membrane fraction if the lysate didn't work. Unfortunately i've only prepared membrane fractions of mammalian cells so you'll have to search for protocols specific to your cell type. But if your game to try the mammalian protocol just let me know and I'll send you what I used.
Dr Washo
[/quote]
The whole cell lysate (and not any particular fraction) is passed through the syringe. The end product will give you protein from both membrane and cell interior.