Hi Gaurav,

sounds you have a contamination in your negative control or your pull down result is a false positive one...

In our pull down experiments I only tried to verify 1 to 1 interactions, so I used the 2 putative interaction partners as recombinant proteins with different tags and one not-interacting protein with the corresponding tag as negative control.

One possibility to get rid of false negative interaction partner could be a pre-clearification of your protein lysate with the beads for 1 hour before the pull down experiment. Another point is to use single Eppendorf cups for your experiment to reduce cross contaminations between experiments.

I transfered my beads with the last washing step into a new cup to get rid of cup-bound proteins - proteins love to bind to plastic columns or Eppendorf cups and you will get them into your sample when you heat your beads in sample buffer for elution.

Good luck!

Dear all, could you send to me your pull down experiments protocol, best.

Hi Gaurav,

There are two possibilities that would lead to a signal in your negative during a pull down (if your negative was carefully pick):

1) your protein of interest interact with the resin or 2) your protein of interest interact with the tag.

Solutions are to change the resin (the dyna beads are magnetic and show little to no unspecific binders) or to change the purification tag (the TAP tag tadem purification steps is the cleanest as it involve a serie of two orthogonal pull downs). A quicker way would be to also adjust the salt concentration in your buffers of the pull down to achieve to a difference in binding between your sample and your negative control (assuming that the protein amounts that binding to the resin or tag do so with lower affinity than the PPI to test).

Good Luck!