Hi,
Is there any way to prove that a protein is membrane anchored/peripheral membrane protein? Any simple trick or any technique will be helpful.
Thanks
Beginning with a total cell lysate containing the protein, separate the membranes from the cytosol by ultracentrifugation (100,000 g for 1 hour). Resuspend the membrane pellet. Test the resuspended membranes and supernatant for the presence of the protein.
Note: do not use detergents for cell lysis because that can dissolve the membranes.
Adam B Shapiro if the lab does not have an ultracentrifuge do you think that filtration through a .22micron syringe filter would work for separating the membranes?
Paul Rutland that would only prove that the protein is not in the aqueous fraction of the sample. Insoluble or very large aggregates of proteins that don't bind membranes won't be able to pass the filter either.
Haaris Ahsan Safdari is your protein already purified or are you wondering whether it binds to membranes in cell or tissue culture or lysate or anything similar?
Arne Raasakka I agree about the cell membranes remaining in the filter and back flushing or dissembling the filter would be needed to access the membranes. I did not know about large proteins not going through the pose and thank you for that information
If you want just a quick estimate, then doing what Adam Shapiro suggests is probably the best mean, coupled to Western blot.
The simple though very slow and labour-intensive way is to overexpress the protein (preferably with a cleavable tag, as the tags will affect membrane association, especially charged tags), then purify it, label it with a fluorescent marker add it onto liposomes containing (different lipid compositions and) a FRET donor or acceptor for the protein label, and follow FRET signal to see if it binds to liposomes. This will not work for proteins that reside at membranes but only by binding to another membrane protein. The added benefit is that you can analyze the binding characteristics.
Alternatively, you could try labelling the protein within cell (by expressing a genetic marker) and try a find a membrane stain FRET pair that has high flip-flop rate and that equilibrates fast between leaflets, so possibly a fairly nonpolar short-chain acyl derivative.
A more interesting option would be to isolate cell membranes, extrude them into small unilamellar vesicles while adding DSPE-PEG-biotin, using silica beads coated with streptavidin/avidin, prepare cell membrane-coated silica beads and use them in chromatography of the cell proteins, collect fractions, then collect fractions with increasing salt, and finally with a detergent. Then you can do a dotblot with the fractions.
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