I want to calculate Kd value for a FITC-conjugated peptide which specifically binds to a cell receptor. I've studied some protocols, but still confused!
Does anyone have any experience or practical protocol for that?
There are two major considerations you should keep in mind in order to avoid introducing error into the assay: The time to equilibrium of the binding reaction and ligand depletion that occurs during the binding assay. That's why a protocol turns confusing and you may found lost on which instruction should be helpful.
On the basis on what you describe here, it seems your trying to determine the Kd under equilibrium-binding conditions and that's what I mostly use. Here you go a protocol for direct binding; I hope this helps.
Aliquot 5×104 cells expressing the target protein of interest to labeled 1.5 mL Eppendorf tubes. Make sure to include tubes for cells only and cells with antibodies only controls, if needed.
Spin down cells at 800×g for 5 min, ensuring cells are still viable by try-pan blue staining. Remove supernatant, and resuspend in binding assay buffer, using appropriate volumes to avoid ligand depletion.
Note: Binding assay buffer can be anything from media to BPBS. All components necessary for the binding interaction of interest should be included (salts, cofactors, etc.).
Add soluble ligand to each tube at varying concentrations, spanning two orders of magnitude above and below the anticipated Kd.
Allow the reaction to incubate at 4°C until it has come to equilibrium, generally a number of hours.
If the ligand is fluorescently labeled, proceed to step 6. If the ligand has an epitope tag, first wash the tubes with 0.5 mL of cold BPBS, and spin them down at 800×g for 5 min at 4°C. Remove the supernatant and then resuspend the cells in 50 μL with a 1:100 dilution of the appropriate fluorescently labeled antibody (i.e., Rabbit Anti-His6–FITC). Allow the antibody to incubate for 20–30 min at 4°C.
Wash cells with 0.5 mL of cold BPBS. Spin down at 800×g for 5 min at 4°C. Remove the supernatant.
Analyze the cells using flow cytometry. Analyze data as described in the yeast binding assay (Postincubation, step 6).
Thank you so much for your useful protocol. I'm going to do it during next few days and will inform you about the results. I searched for "yeast binding assays"(as you mentioned in step 7) and found some papers. It would be very kind of you if you could explain more if there is any practical point with this assay.