How to demonstrate protein-protein interaction - How to demonstrate that a protein is a receptor (May/10/2008 )
Hello. Thank you for reading. I'm a first year graduate student. My question is how to demonstrate protein-protein interaction. Actually, my real objective is to know whether my protein of interest acts a receptor. I'm planning to do co-immunoprecipitation and confocal microscopy, but I'm not sure whether these methods are appropriate and enough to meet my goal. Your suggestions and comments will be highly appreciated. Thank you
the more different methods to show protein-protein interaction you use the better; your suggested methods (Co-IP, CLSM) are weak but commonly accepted methods to demonstrate protein-protein interaction;
my question is if you know your binding partners or are searching for them?
You being a first year grad, I think you must read a review of vast array of methods available these days for p-p interactions, such as Co-IP, Yeast two-hybrid and a lot of variations on it, FRAP/FRET, SPR, SLS, DPI, SPINE, BiFC etc.
This may sound like no help, but that is the best way to go.
the more different methods to show protein-protein interaction you use the better; your suggested methods (Co-IP, CLSM) are weak but commonly accepted methods to demonstrate protein-protein interaction;
my question is if you know your binding partners or are searching for them?
Thanks. From literature I found one protein that binds to my protein of interest, but my goal is to find other binding partners. What other methods can you suggests? Especially in demonstrating that proteins act as receptors.
This may sound like no help, but that is the best way to go.
Thanks. I'll check out the methods you wrote.
the more different methods to show protein-protein interaction you use the better; your suggested methods (Co-IP, CLSM) are weak but commonly accepted methods to demonstrate protein-protein interaction;
my question is if you know your binding partners or are searching for them?
Thanks. From literature I found one protein that binds to my protein of interest, but my goal is to find other binding partners. What other methods can you suggests? Especially in demonstrating that proteins act as receptors.
some try by Co-IP with subsequent Ms/Ms; however, you may identify dozens of co-immunoprecipitated proteins; microscopical methods are not useful at this stage...
There are stringent requirements to determine if a protein acts as a receptor. You must do in vitro binding studies with the ligand, determine binding constants, stereospecificity, etc, and when all is complete you will probably need to show that the binding is physiologically relevant (i.e. loss of the receptor produces a phenotype that one would expect for the function of the protein). Also keep in mind that not all receptors are membrane proteins. I would recommend that you look up some papers on receptors particular to your topic to see what has been done in the past.
thank you for the suggestions. My protein of interest is relatively new. I've done my research and found only 3 papers on my protein. From their discussions, I thought that the protein may also act as a receptor that's why I suggested this topic to my supervisor. I'm trying to look at papers of other proteins to see if I can also do what they did to demonstrate one of my objectives. thanks again.
thanks. co-ip, sds-page, wb, sl, and ms were the methods i originally submitted to my supervisor. i was also planning to do 2-hybrid system, but he said i will just get the same results with co-ip. however, i feel that my methods are still not enough to meet my objectives, or at least to write a paper that can be published.
Biacore. ITC.