FAQ

Q:  We will be using your chick-ZAP secondary conjugate (Cat. #IT-62) and noticed that in your protocol you mention not to use a reducing agent in your media. Our normal growth media contains beta mercaptoethanol at 100 μM. Will this be a problem?

A: Officially, we would recommend allowing the cells to acclimate to media that contains NO BMe, and then proceed with your experiments. However, some of our in-house experiments use cells that are cultured in media containing 50 μM BMe, and we have not seen that concentration affect the toxin’s effectiveness, but we have not tried a concentration as high as 100 μM.

Related Product: Chick-ZAP (Cat. #IT-62)

Q: We are interested in having a custom conjugation of saporin and our antibody. Do you remove unconjugated antibody from the final material you send us?

A: We do remove the unconjugated antibody and saporin from the final product that we send to you. And perhaps to answer a question you may not be asking, but may be curious about, the unconjugated material is not particularly usable, after it has been removed from the final product as it has been slightly modified in preparation for the conjugation.

See also: Custom Conjugates

Q:  I’d like to know which of your products are the pan-neural targeting toxins? I need an agent to kill all nerves in tissue preps.

A: OX7-SAP (Cat. #IT-02) should be perfect for this application. We recommend you examine your neurons with OX-7 antibody to see if they are positive. The only complication would be if you want to look at T-lymphocytes that also express Thy 1.

Related Product: OX7-SAP (Cat. #IT-02)

Q: I plan to use your Secondary Antibody Conjugates, Rab-ZAP (Cat. #IT-05), and Fab-ZAP Rabbit (Cat. #IT-57) with my primary antibody and would like to observe eliminated cells using a fluorescence microscope. The idea is to co-culture cancer cells and fibroblast cells, and kill fibroblast cells only with a specific primary antibody. Then I want to observe the eliminated fibroblast cells and take pictures with a fluorescence microscope. Can you recommend a protocol?

A: In order to stain and visualize the cells that are being eliminated, it would be best to stain for Saporin using a fluorescently-tagged antibody such as the FITC-labeled Saporin antibody (Cat. #AB-15AP-FL). By washing off the media after a day, and then staining for saporin, one would illuminate only the cells that have internalized the saporin (marking them for death). The cells that do not stain for saporin will live.

Related Product: FITC-labeled Saporin antibody (Cat. #AB-15AP-FL)

Q: I have a question regarding your antibody to NGF (p75) receptor antibody (Cat. #AB- N01AP). Could you please tell me how you determined that it is a blocking antibody? Has this information been published?

A: We list on our website that one application for this antibody is for blocking the function of nerve growth factor receptor. This information was presented in an abstract at the Society for Neuroscience Meeting held in 1994.

Huber LJ, Lee K-F, Dreyfus CF, Chao MV (1994) Generation and characterization of a murine p75 receptor blocking antibody. Soc Neurosci Mtg, Miami Beach FL, Abstract #23-12.

Please check out the other references  on our website for publications describing applications for this antibody.

Related Product: Anti-NGFr (Cat. #AB-N01AP)

Q: I have a mouse monoclonal antibody and a rabbit polyclonal antibody that I would like to test using your secondary conjugate system. Which products do I need to order? 

A: For mouse monoclonals, you can use: Mab-ZAP (Cat. #IT-04) – Cells that internalize your mouse monoclonal antibody will be eliminated. Or, Fab-ZAP mouse (Cat. #IT-48) – Cells that internalize your mouse monoclonal IgG antibody will be eliminated.

For rabbit polyclonals, you can use: Rab-ZAP (Cat. #IT-05) – Cells that internalize your rabbit polyclonal antibody will be eliminated. Or, Fab-ZAP rabbit (Cat. #IT-57) – Cells that internalize your rabbit IgG antibody will be eliminated.

The difference between Fab-ZAP products and other secondary conjugates is that Fab-ZAP is made with a monovalent secondary antibody which eliminates the possibility of cap formation as cross-linking of the Fab-ZAP molecules cannot occur. The Fab-ZAP products will still recognize the heavy and light chains of antibodies, and should be used in the same way and at the same molar concentrations as the original secondary conjugates (such as Mab-ZAP and Rab-ZAP). Cytotoxicity assays using Fab-ZAP (mouse) have demonstrated an improved EC50 when directly compared to Mab-ZAP.

Any of our secondary conjugates offer a very cost-effective diagnostic method for screening primary antibodies for in vitro or in vivo use.

Related Products: ZAP Conjugates

Q: 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?

A: 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.

Protocols

Q: 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?

A: 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.

Related Products: Bombesin-SAP (Cat. #IT-40), Blank-SAP (Cat. #IT-21)

Q: 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?

A: The effect you are seeing is something that is actually typical and indicates that you are using the material correctly. Described in Kohls et al., unbound primary antibody will compete with primary antibody bound to a secondary conjugate 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.

Related Product: Mab-ZAP (Cat. #IT-04)

References

1. Kohls MD et al. Mab-ZAP: A tool for evaluating antibody efficacy for use in an immunotoxin. BioTechniques 28(1):162-165, 2000.

Q: 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?

A: Mab-ZAP will recognize whole IgG and will bind to both the heavy and light chain.

Related Product: Mab-ZAP (Cat. #IT-04)