Regarding Secondary Conjugates
I would like to know if the secondary antibody used to prepare Mab-ZAP (Cat. #IT-04) reagent binds to heavy chain of mIgG’s (only) or if it recognizes light chains as well?
Mab-ZAP will recognize whole IgG and will bind to both the heavy and light chain.
We’ve been using Mab-ZAP to test our primary mouse monoclonal antibody in cytotoxicity assay. Relative to the Primary mAb-Mab-ZAP complex, the Mab-ZAP alone has quite a bit of activity (40-60% growth inhibition) at the recommended concentrations used (45 or 100 ng/well) or even half that dose. Even though we get a dose response, the higher concentrations of primary antibody give me less cytotoxic activity than the lower concentrations. Can you give your thoughts on this matter?
The effect you are seeing is something that is actually typical and indicates that you are using the material correctly. Described in Kohls MD, Lappi DA (2000) BioTechniques 28(1):162-165, unbound primary antibody will compete with primary antibody bound to a secondary conjugate and may reduce cytotoxicity through competitive inhibition of the primary antibody-Mab-ZAP complex. This is especially noticeable with our Fab-ZAP line of secondary conjugates.
We still recommend that our customers try a 10 nM dose as a starting point, but always recommend adjusting the concentration to better accommodate their experiments. As a reference, our data sheets show a cytotoxicity curve with a starting concentration of 10 nM and an ending concentration of 1 fM.
See also: Secondary Conjugates Catalog
Regarding Other Products
I am using your Bombesin-SAP (Cat. #IT-40) to kill GRP-receptor in mouse brain. I have a plan to inject it by iontophoresis. Do you know which charge dose Bombesin-SAP and Blank-SAP (Cat. #IT-21) have; plus charge or negative charge?
Both of these products will have a negative charge, though you may have to look into the literature for any needed guidance on dosing with that type of delivery.
We are setting up some experiments in rat where we’d use your 192-IgG-SAP (Cat. #IT-01) in cytotoxicity assays. How much material do we need to order?
You can find protocols for calculating the amount of material needed for a cytotoxicity assay and protocols for the assay and interpretation of results on our website. Just click on the PROTOCOLS link on our home page.