Hello, I am a intern, very new to pull down assays. I am planning a His- pull down experiment. I have His tagged Protein A and an untagged protein B. I want to know whether they interact each other in presence of a drug. The drug concentration at 10uM/10ul worked well in our cell line assays. How do I go about this? Theoretically, I need a drug only and drug naive conditions to test.Basically I plan to bind purified His protein and untagged protein (5-10ug per reaction) to dyna his beads 1 - 2hrs at 4 degrees with and without drug
in binding buffer). Wash beads 4X in wash buffer and do SDS page. Do I need to dilute the proteins in the binding buffer? If 10uM final concentration of drug worked in cell lines, how much drug do I need for the pull down assays? Please help me