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As you requested, here is the feedback from the replacement antibody for PERP (ab5986). Please see attached image of WB, obtained with 1 ug/ml of Ab (ab5986), standard conditions, as described in my previous message (regarding use of the other PERP Ab - ab 3945). M=size markers, as indicated; A431 cell lysate, as positive control; 1-4 our samples. Equal total protein loading in all lanes, as demonstrated by GAPDH probing. ab 5986 appears to recognize a band of the expected size, i.e. 21kDa, which was not recognized by the other Ab. However, note that two other bands appear, with the 37 kDa one being the band which appeared strongly immunopositive with the ab 3945, as described previously. Your comments regarding this will be appreciated. Best regards, Luminita.
Asked on Sep 28 2004
Thank you for getting back to us and providing us some more information about your recent results. We can see on the Western blot image that the 21 kDa band appears nicely, however there are two other non-specific bands as well. It is important to emphasize that ab5986 is a polyclonal antibody and it recognizes different epitopes. Therefore the antibody may recognize other proteins as well which share similar amino acid sequences. We would suggest perhaps reducing the concentration of the primary antibody and applying more stringent washings. Have you tested the secondary antibody alone? We hope this will help.
Answered on Sep 30 2004