Hello Laura, if I understand you correctly, you incubated lysed cells with the beads before checking for expression of your protein? I would start over for a few reasons. One, it is always best to purify your protein as fast as possible after thawing and lysing the cells. Two, and related, there are endogenous proteases that will likely chew up your sample. Three, your protein may denature over time. Four, non-specific absorption of endogenous proteins to the beads will increase over time. You can just boil a small aliquot of the beads and check what is there via SDS-PAGE. Were it me, I would just start afresh. Good luck!

Hi Jamison, thanks for your answer!

Actually, I didn't incubated lysed cells with the beads. I'm using isolated GST-fusion proteins that are already purified from bacteria. I'm using them as a bait. The goal is to mix them with mammalian cell lysate to see whether there is an interaction. So, before lysing my mammalian cells, I have already coupled the GST-proteins to the GST-beads. So, I haven't gotten to the point of using my lysates.

Still, it does make sense that the proteins might have suffered upon thawing and staying in the fridge for too long. I will do it as you suggest - thanks!

Laura, ah ok thank you for the clarification! Well you could try but the danger is that if the pull-down does not work then you do not know if it is because there is nothing to pull down or because the protein denatured. If there is a known interacting protein that would be good as a positive control. So yes, fresh is best. Good luck!