Our lab is trying to establish a method to detect the interaction between two proteins. There are several ways to do this, among which the AlphaScreen assay or a co-immunoprecipitation assay.
We've had some problems with the AlphaScreen in the past when we used recombinant GST-, MBP- and His-tagged proteins. We haven't done a Co-IP yet.
Which one do you think is the best/easiest to use? And why?
Hi Art,
Well all techniques have pros and cons. I don't know much about SPR and BLI that look very powerful to characterize an interaction with a measure of KD.
I'm not a fan of fusion proteins to evaluate a biochemical interaction between 2 proteins because GST, GFP or MBP are quite big and can change a lot of things in terms of ternary (and quaternary) structure. I rather used such fusions, especially MBP to have them immobilized on beads for affinity-purification of antibodies from serum.
If you already have GST fusion you also can consider GST-pull down. You immobilize your GST fusion on beads and apply a lysate of your cells/tissue of interest on the column and check by WB if your protein of interest "sticked" to it.
Regarding the co-IP i'd consider try it with small (8-10 AA) tags like FLAG, HA, myc on the 2 proteins of interest. If you don't have the constructs you can possibly quickley get them on addgene, transfect HEK cells and do the IP with Flag M2 beads and WB with anti-the chosen tags.
The limitations of this are to my point of view the following:
- you need a lot of controls like IP the same protein with different tags/or other proteins with Flag tags to make sure it is not creating the interaction
- Setup of wash NaCl conc. to keep specific interactions while getting rid of non specific.
- it is overexpression hence source of artifacts and if you are asked to validate it on the endogenous protein is another story (still feasible but difficult)
- The anti-tags antibodies can be dirty for WB so it is better to have a good antibody directly against the proteins of interest to verify consistent IP of the first protein across the conditions and co-IP or not of the second
- There will be a lot of IgG heavy and light chains on the WB membranes so parasite bands at 50 and 25 kDa if you can't to else than IP and WB with Abs made in the same species.
Good luck
Daniel
It depends on your study, look at plasmon resonance and thermal shift assays for protein-protein interaction
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