minimum recombinant protein amounts for gst pulldown - (Jan/15/2013 )

Dear All

I have already observed interaction of 2 proteins using co-immunoprecipitation methods from total cell lysates.

I would like to check whether this interaction is direct. Therefore, I decided to use recombinant proteins (one protein with GST tag) and make a GST pull-down. The problem is, we are not very experienced with protein purification so decided to buy the recombinant proteins, which are quite expensive.

Do you have any suggestions on how much protein is enough for such a pull down? I bought 10 micrograms each only. When I check papers they always use 10-50ug for each pulldown, but I am guessing this is only because they purified the protein themselves and used a lot of it. Is it enough to use about 1ug each for an experiment?

Thanks in advance!

Hi,

during my master I've been using 1-2 ug protein (purified by me) with GST tag for pull-down. But I've been using 20 ul Glutathione Sepharose not ready kit for co-percipitation... I don't know how it will be work with your protein (you need to check conditions ), but in my opinion you can try .
Ewa

Thanks Ewa!
I also want to use Glutathione beads. I thought 20-30ul would be enough and then I was planing to boil the beads in equal amount of laemli buffer after washing.

I can sent you my protocol if you want

That would be really great!