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I apologize for the confusion, but from previous emails I did not mention that we performed any staining experiemtns on the BD-3 antobody. To clarify things, a total of three experiments were conducted on BD-3(ab14422) antibody. First, the BD-3 (ab14422) mouse monoclonal antibody was coated onto an immunoplate. We used an anti-mouse and an anti-rabbit IgG as a secondary antibody to probe your antibody. The results from this experiemtn indicated that there was a 100% corss reactivity with the anit-rabbit IgG secondary antibody. As previously mentioned, these secondary antibodies are widely commercialized. We have used these antibodies many times and have not found them to cross react with other species. Second, a goat anti-rabbit coated immunoplate was used to probe the BD-3 (ab14422) antibody. We used an anti-rabbit secondary anti-rabbit antibody to probe the BD-3 again. The results from this experiment showed that were was still binding for the rabbit. Third, we coated an immuniplate with BD-3 peptide and used BD-3 (ab14422) and a control antibody were used as primary antibodies. As expeted the control showed binding while the BD-3 antibody (ab14422) did not. If you still require the data, I can send it as well.
Asked on Oct 17 2011
Thank you for the clarification. I have just a few more questions. 1. In the first experiment, did the anti-mouse secondary react with ab14422? 2. What is the source and catalogue number for the anti-rabbit secondary that reacted with ab14422? 3. For your second experiment, did you test in parallel any other mouse IgG, in addition to ab14422? What is the source and catalogue numbers of the two anti-rabbit antibodies? I understand you have not seen this before with your anti-rabbit secondaries, but polyclonal secondaries can vary lot to lot with regards to species reactivity. 4. In the third experiment, was the peptide that the plate was coated with the same as the peptide used as the immunogen for ab14422, LQKYYCRVRGGRCAVLS, corresponding to amino acids 6-22 of Human beta 3 Defensin? What is the control antibody in this experiment? What is the secondary? We will not need the data, as you have described the issue well. At this point, I still suspect the specificity of your anti-rabbit secondaries. On the otther hand, if the peptide coated onto the plate contains the ab14422 immunogen sequence, and the anit-mouse IgG secondary fails to detect ab14422, and is not isotype subclass-specific (for example, anti- IgG2a), then I do suspect ab14422. I look forward to your reply.
Answered on Oct 17 2011