extraction of membrane protiens - protocol for extraction of membrane bound protein? (Sep/07/2005 )

can someone recommend a protocol for the extraction of membrane bound protiens from a cell membrane - my target protein is a channel on the pancreatic beta cell!!

any help would be much appreciated! i am lost!

-janbrisbane-

I have the similar problem as you. I am trying a membrane protein extraction Kit from BioVision. But it did not work well to my samples. If you do not mind, you may try it. My problem is my cell lysate is not well homogenized.

Good Luck!

QUOTE (janbrisbane @ Sep 8 2005, 12:53 AM)

-Kevin_1999-

I also have the problem. I want to extract golgi apparatus bound proteins without affecting its activity because I am going to do enzymatic reaction with the cell lysates. I tried detergent but not working. Any suggestions will be highly appreciated.

Lana

-lanban-

There is a commerical Solubilizer which is specially used for "hard to dissolve" protein.
Please find the details from the following link.
http://www.invitrogen.com/content/sfs/manu...bilizer_man.pdf

I have never tried it personally. You might probably need to try it out.

-sallylyc-

Any of the myriad detergent based lysis buffers should give you a fine start. Something that contains deoxycholic acid and perhaps some tween and SDS that is isotonic is what I would recommend. Your biggest issue is temperature. Membrane bound proteins can be a big problem to get in monomer form. My recommendation is to play with temperature but I'd bet that any warming of your sample above room temp at any stage is going to give you problems. Good luck.

-ShawnB-

QUOTE (janbrisbane @ Sep 8 2005, 02:53 AM)

You may want to look at a paper by Michael C. Wiener in Methods 34(2004) 364-372.

-mjray-

i am kind of glad to hear that i am not the only one stuck!

thanks for that paper's referenc e- it looks promising! i'll try it and let the rest of you know how that went...

-janbrisbane-