I am isolating plasma membrane from a neuronal cell line. Currently my protocol steps are as follows:
1- scrape and spin cells down at 450g for 10min.
2- Sonicate pellet in buffer and centrifuge at 12,000g for 20min.
3- collect supernatant and centrifuge at 50,000g for 1hr.
4- remove supernatant and homogenize pellet (the pellet is the plasma membrane fraction)
So far I have detected Na/K ATPase (PM marker), but I have also detected calreticulin (ER marker) and Rab5 (late and recycling endosome marker) indicating contamination of other organelles. I was considering doing an additional separation on 30% percoll with the remaining PM-containing pellet at 84,000g for 30min and collecting the highest band. Does anyone have a protocol they could provide or a next step recommendation based on their own experience?
In a previous project we made use of disruption of cells by nitrogen cavitation (Cold Spring Harb Protoc. 2010 Nov 1;2010(11):pdb.prot5513. doi: 10.1101/pdb.prot5513). The contamination with other organelles was reduced but nonetheless detectable.
In the past, I was interested in the purification procedure. Maybe you'll find this .ppt presentation useful. (attached)
I have considered biotinylation as well, thank you for the recommendation.
Dr. Kerkhoff I will read into that protocol, as it could certainly be a useful protocol for my purposes.
Dr. Stipčević this is a very helpful PowerPoint, especially for determining proper reagents in extracting my membrane protein of interest.
Thank you all!
There is a Plasma Membrane Protein Extraction Kit from Abcam (ab65400). They also have a detailed protocol. I hope it will help.
You could find great protocol for mentioned task here http://www.nature.com/nprot/journal/v9/n2/full/nprot.2014.016.html
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