Q: For in vitro cytotoxicity assays, could you tell me: 1) whether you incubate primary with your Saporin secondary for a specific amount of time prior to cell addition, and 2) do you use a single concentration of secondary per well or a primary:secondary ratio — like 1:2 or 1:4?
A: The primary antibody should be incubated with the ZAP product for 20 min prior to addition to the cells. Internalization often happens so quickly that you would lose some efficacy due to the antibody being bound and internalized prior to the ZAP product complexing with the primary. We do recommend maintaining a constant 5 nM (~ 45 ng/well) concentration of the ZAP product in the well and titrating your primary only. This way the EC50 you generate will be the EC50 of the primary antibody with all else held constant. The best starting concentration for your primary antibody is 10-100 nM in the well.