I am trying to express protein on COS-7 cell membrane with a GFP tag. Can any one help me out on how can I purify the membrane protein for western analysis and may be even for flow cytometric analysis.
The protein is expressed in its native form with out any purification tag (his or other for that matter).
Any help in this will be appreciated.
If you can isolate the plasma membrane fraction, you can then solubilize the membrane and IP the protein with anti-GFP agarose beads (the ones from MBL are very good, D153-8). alternatively, you could put a BirA tag on your protein, fractionate, treat with BirA to biotinylate, and then purify with streptavidin, etc.
If the expression levels are high enough, there is no need to purify membrane proteins for western blot. If you are concerned about wild type protein, as your protein is fused to GFP it will migrate slower in SDS-page (Check its theorical MW). Besides, if protein fusion did no alter GFP fluorescence, you can analyze your cells by FACS.
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