Protocol Online logo
Top : Forum Archives: : Protein and Proteomics

GST and Yeast Hybrid Assay - (Apr/15/2008 )

Whats the difference between the Yeast Hybrid assay and GST pull down assay, while both are used for the interaction.


If you are trying to prove two known proteins interact, either one can be used.

If you are trying to search for new interacting proteins, the 2 hybrid system will give you gene identity quickly (but it is usually false positive) and the GST pull-down assay will require that you identify the interacting proteins by other techniques (western blots being the simplest, if you know what to probe for). Both assays will be skewed towards the most abundant proteins.

I'd be wary of undertaking a 2 hyb project to identify interacting proteins. You can end up with more questions than answers. It is very hard to prove an interaction is relevant, especially if you identify a protein that nothing is known about and you can't readily purchase an antibody to rapidly confirm the interaction by another method (co-IP).



I'm using Y2H assays for library screens and basically for checking specific interactions between two known proteins. This is relatively easy to do compared to specific pulldown assays where you have to purify your proteins and coIPs (since I'm working with proteins which make only very weak interactions). But anyway, If you find an interaction in Y2H it looks always good in publications if you can prove your interaction with a second independent assay.