Hi everyone, recently, I am going to study two protein interaction by GST pull-down. I have protein GST-A and MBP-his-B expressed by BL21 through IPTG induction. Initially, I found GST-A can pull down MBP-his-B (only GST can not) so I decided to further construct the plasmids which express different domains of protein A fused with GST. Then I performed GST pull down and the result is that all domains can pull down MBP-his-B and this conflicts with my hypothesis (my hypothesis is only three domains of A can interact with B). So I think this could be false positive (although my control GST can not pull down B). Can anyone give me some advices on how to reduce the false positives in GST pull-down?
I used 1% tritonx-100 in PBS (with 1% proteinase inhibitor cocktail) and sonication to lyse bacteria. I used sepharose 4B beads to bind GST fusion protein at 4°C for 1 hour. After wash three times with 1% tritonx-100 in PBS, I incubate the beads with bacteria lysate containing MBP-his-B at 4°C overnight.
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