I am interested in investigating the ability of a certain protein to associate with cell membranes. I have some ideas but I'm sure that I haven't thought of every possible technique. In the past I have used an ELISA-based method with lipid-coated 96-well plates, but I'd like to have multiple lines of evidence to really nail down this interaction.
There are two other methods I'm considering:
1. pull-downs with streptavidin-immobilized biotinylated lipsomes
2. EPR/site-directed spin labeling (see Malmberg et al. DOI: 10.1146/annurev.biophys.34.040204.144534)
Any other suggestions??
Thanks in advance!
Western blot with membranes separated from whole cell extracts by ultracentrifugation.
Are you interested in the thermodynamics of the interaction, the binding site, the effect the protein has on the membrane, the effect of lipid composition on binding, or something else?
Is this a purified recombinant protein or a cell extract?
Hi Justin,
maybe it is stupid or you already used the method of alkaline treatment to remove peripheral proteins from a membrane. I remember that a colleague of mine used this method to analyze peripheral membrane binding of proteins. I don't know If you have an antibody against your protein of interest or a tagged form, but it would be possible to design an experiment like add "protein" to an artificial membrane or maybe something like trypsin-treated cells (to shed most of the proteins from the membrane) and do an alkaline treatment, neutralize, and wash, wash, wash. Then do the the alkaline treatment again and do an WB (with something like a LiCor or similar to be a bit more quantitative).
Here is the link to a description of the method (quite long, is from a book)
https://books.google.de/books?id=_wibShTmB5IC&pg=PA284&lpg=PA284&dq=alkaline+treatment+membrane+proteins&source=bl&ots=wWtcw9OUM_&sig=PYToH10z7vyBCiJ8BR0AIu0ygSk&hl=de&sa=X&ved=0ahUKEwjP_siQ2aHLAhULlSwKHWvoAEIQ6AEIRzAF#v=onepage&q=alkaline%20treatment%20membrane%20proteins&f=false
Good luck,
Carsten
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