I am currently doing the GST pull down using a few GST-fusion proteins as bait to confirm protein interaction with my GST fusions. as you all know the experiment theory is to compare prey output between GST control and GST-bait. The question is what is the best way to make sure the GST and GST-fusion are same molar. Many thanks,
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GST pull down
1 reply to this topic
Posted 05 May 2010 - 10:59 AM
Generally, it's acceptable for the amounts of GST (on glut beads) and GST-fusion protein (also on the beads) to be roughly equal as observed on a ponceau stained membrane before you probe it. Before you do the real experiment, run all your GST preps on a gel, coomasie stain it, and roughly quantitate the proper-sized band (there might be degradations products... these are not always a problem). When it comes to the experiment, if the GST-only control is visibly equal to or greater than your fusion protein, people will accept the conclusions. You should see NO binding to the GST-only control if the experiment is done well. You can test different quantities of GST proteins or lysates...etc. to fine tune the assay. Temperature, time, and pH might matter as well.