Regarding Custom Saporin Conjugations
We recently spoke to you about performing a custom saporin conjugation (see Custom Conjugates) using our antibody. Is 0.09% azide in PBS in the antibody stock acceptable?
There are a number of dialysis steps within the conjugation protocol that will ultimately remove the azide from your antibody solution. So as long as your antibody will be happy in PBS without azide during the procedure, sending the material in 0.09% azide is fine. The final conjugate will be returned to you in PBS, sterile-filtered, without azide.
In general, how many saporin molecules are incorporated per antibody? Can we test this by HPLC?
We aim for 2-2.5 moles of saporin per mole of antibody. You should be able to see differences in HPLC between your antibody with one vs. two vs. three saporins attached, however we will provide you with a saporin molar ratio and a product that has had free saporin and free antibody removed from the final conjugate.
Regarding Quinolinic Acid Antibody
We need to have quinolinic acid conjugated to BSA to coat our plates for our experiment. Do you have a protocol to perform this conjugation? We have already purchased your Quinolinic Acid Mouse Monoclonal (Cat. #AB-T170).
We can do better than that. You can purchase quinolinic acid pre-conjugated to BSA (Cat. #AG-033).
Regarding Assay Parameters for Mab-ZAP
Using Mab-ZAP (Cat. #IT-04) in a cytotoxicity assay, I obtained a nice dose-response curve up to around 10-9 M of antibody and then lost progressively the toxic effect of Mab-ZAP. What should I do to improve in my assay?
If the highest dose for which you got a good response was 10 nM and you lost effect when the primary concentration was increased beyond that, then that is the result we would expect. Often we have seen in cytotoxicity assays that when the primary antibody concentration is raised beyond a certain level — 10-100 nM frequently being that level — there is so much free primary antibody that it competes with the Mab-ZAP-bound antibody for binding sites, thereby reducing the toxic effect. We recommend that you pre-incubate your primary with Mab-ZAP before adding the solution to the wells.