Question (12761) | Anti-Notch1 intracellular domain antibody (ab8387)

Go to datasheet (ab8387)

Question

I bought anti-Notch1intra antibody (product n. ab8387) and tried it but it did not function well, too many bands and there was a band also at the right molecular weight in a mouse extract which should not be, because the antibody, as says the data sheet, should not cross-react with mouse. I used a classical protocol (5%milk in PBS with 0.2%Tween-20 which should be more stringent than BSA). So my question is: Do you have a picture of an experiment with Notch intra at the correct molecular weight made on human and mouse extracts in order that I can see what I should expect? Another question: how is it posible that this antibody recognizes only the Notch intra domain, since the longer forms of that protein should all contain the epitope at aa2788-2502?

Answer

Thank you again for your email and patience while I have been trying to obtain additional information regarding this antibody. Unfortunately, there is no additional information regarding the testing of antibody, including a Western blot image. A colleague and I compared the human and mouse Notch1 intra sequences (amino acids 2488-2502) and there is 93% homology between the two sequences. Also, the epitope recognized by this antibody may be exposed in the cleaved form, but not in the full length form, even though the sequence is found in both. What dilution of ab8387 are you using? What is your secondary antibody and what dilution are you using? Have you done a secondary-only control to ensure that the secondary is not binding non-specifically? Also, what is your order number? Please rest assured that we guarantee our antibodies to work as stated on the online datasheets. I will do my best to troubleshoot this problem, and can offer you ca refund or replacement. Thanks you again, and I look forward to hearing from you.

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