Membrane protein extraction from mice tissue - (Jan/07/2008 )
I need to isolate IFNAR1 (interferon receptor- 11kDa) from mice tissues (spleen, thymus, lymphnodes etc.), in order to do a Western Blot. This is a membrane protein of course and I haven't done extraction of such protein before.
Would you please suggest me a protocol to try?
Thanks a lot!
Simply homogenize the tissue samples in 1X SDS-PAGE sample buffer with SDS should do.
As already suggested, SDS buffer would extract the membrane proteins.
What about that protocol? It was suggested to -vvmice- by -Chakchel-...
2 ml 1M Tris-Cl pH 7,4
500 µl 1M MgCl2
200 µl 0.5M EDTA
120 µl 0.5M EGTA
ad 100ml Aqua bidest.
You can store this buffer at 4°C until you need it.
Before use I add one tablet of Complete (Roche) to 10 ml to inhibit proteinases.
For extraction I put some lysis buffer to my washed cells, collect them by scraping and then break the cells using a insuline syringe. Then I centrifugate the suspension for 5 minutes (200xg, 4°C) and take the supernatant for another zentrifugation (30 min, 43,000xg, 4°C). The pellet I gain contains the membrane proteins. I usually resuspend it in 100 µl lysis buffer.
It seems more confusing than a simple SDS buffer.....