Anti-Diphtheria Toxin抗体(ab53828)

Thank you very much for your reply.

I am sorry that these suggestions are not more helpful. If you would like to try a different DT antibody please let me know and I can arrange a testing discount for you.

If there is anything else that we can do for you, please don't hesitate to ask and I'll be happy to assist you.

Thank you very much for your reply and for sending this additional protocol information and the images.

I've looked through the images and alsodiscussed the situation with some of my colleagues. Based on these images, it looks like the antibody volume was held constant with increasing amounts of protein. All of the mouse antibody ab51882 seems to be binding to the beads, but the protein to antibody ratio needs to be optimized. We would recommend using a minimal amount of protein (1 ug or less) and holding this quantity constant while increasing the volume of the antibody, in order to find the optimal protein:antibody ratio. By using a higher quantity of purified protein, the antibody may be saturated so that it can not bind any more protein.

With the rabbit antibody ab53828 (I mis-typed ab53848 in my previous email), it looks like there may be too much protein loaded onto the gel. I'd suggest a similar protocol with a small amount of protein held constant with increasing amount of antibody. It's possible that the rabbit antibody has a higher binding affinity compared to the mouse antibody, and less of this antibody can be used.

As I mentioned before, we haven't tested any of our DT antibodies in IP so we aren't sure which will be most suitable for this application. If you would like to try one of our other DT antibodies, we do have a testing discount program that you can participate in. I might recommend the goat polyclonal ab19950 instead of one of the other monoclonals, as the polyclonal antibodies will have more binding sites on the protein.

I hope that this will be useful, but please keep me updated of any progress and let me know if you have any questions or if there is anything else that we can do for you, and I'll be happy to help.

Thank you very much for contacting us with your questions and for sending the details of your protocol.

I do have a few additional questions just to make sure that I understand the situation:

1) How is the CRM197 prepared prior to the IP? Is it in a denatured conformation?
2) In what buffer is the CRM197 incubated with the antibody? What is the total volume during this incubation (protein, antibody, and buffer)?
3) Is there a reason why the elution was done at 100C for 5 min instead of 50C for 10 min? Have you also tried a double elution?
4) Do you have any images of your results?
5) Have you tried using protein A antibodies with the rabbit polyclonal ab53848 (slightly higher affinity than protein G)?
6) Have the antibodies been tested against the CRM197 protein in a Western blot or ELISA?

We have only tested these antibodies in Western blot and ELISA, so we don't have a specifically optimized IP protocol for them, but I may have some suggestions after looking over the additional details. It is also possible that these antibodies are not suitable for use in IP. Unfortunatley none of our DT antibodies have been tested in IP so we don't know which would be best to use in your assay, however we do have a testing discount program available if you would like to try a differentDT antibody in IP.

I look forward to hearing from you. Please let me know if you have any further questions and I'll be happy to help.