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GST pulldown: elude or not? - (Dec/13/2010 )

Hi there,

I'm wondering, what most people do for GST pull-down experiments. I've read a number of protocols and know people who never bother to quantify exactly the GST protein and just load the bacterial lysate to the beads, wash and then load the second lysate (with His or GFP tagged protein) to the same beads, incubate and boil.

On the other hand, some quantify very exactly the amount of the first and second protein, etc.

I see possible strengths in both options. In the first instance, one doesn't loose any protein in elution and doesn't care about possible aggregation in elution buffer or protein storage. For rapid experiments one saves a bit of time. On the other hand one faces a risk of overloading the beads with a protein and getting false positive results? So I am curious what is the prevailing method?


and a second question to complement the first:

in case you quantify the amount of prey and bait protein, how much do you take for one reaction?