Hi everybody,
I need to extract internal-associated membrane proteins from mammalian cell lines, eventually without using ultracentrifuge. My aim is to analyze protein expression by western-blotting.
Best regards
Dear Alessio,
I attached a protocol I have used, from Dr. Brian Wattenberg's lab.
It has was for a lipid based radioactive kinase assay for studys with human sphingosine kinase.
You can just use the first half for the harvesting of mem. and discard the labeling and transport assay
As I recall the resuspending of the membrane pellet was not easy and took time.
Best of Luck!
We are doing membrane protein extraction by biotinylation and avidin pull-down. Good for WB. You can try milder lysis buffer and cell lysis method with this.
For protein extraction you can use one of these:
- http://www.bio-rad.com/en-cn/product/readyprep-protein-extraction-kit-membrane-i
- http://www.bio-rad.com/en-ch/product/readyprep-protein-extraction-kit-membrane-ii
I have used cell lines and primary culture of human brain and brain tumors for whole cell protein isolation and I have used normal tris buffer with triton 1%. But for membrane proteins density gradient centrifugation / ultracentrifugation will be fine to proceed for protein isolation.
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