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BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 167107 DESCRIPTION OF THE PROBLEM no band SAMPLE Cell extract PRIMARY ANTIBODY ABCAM/Rabbit/1:1000 and 1:500 dilutions in 5% milk/TBST(Also tried without blocking with milk at all---using TBST alone. overnight incubation DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED ABCAM Beta actin purchased last year used as negative control. ANTIBODY STORAGE CONDITIONS -20 degrees SAMPLE PREPARATION RIPA buffer/PMSF AMOUNT OF PROTEIN LOADED 30 micrograms ELECTROPHORESIS/GEL CONDITIONS 10% gel (acrylamide gel) TRANSFER AND BLOCKING CONDITIONS Transfer buffer: Tris base 48mM--5.8g, Glycine 39mM---2.9g, SDS 0.37g, 20% methanol---200ml to make 1L. Blocking agent: 5% milk. Blocking time: I hour SECONDARY ANTIBODY Santa Cruz/Rabbit/5% milk/TBST/1:1000 Incubation for 3 hours at room temperature HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? No
Asked on Sep 18 2006
I'm sorry to hear you are having a problem with ab4830. I did have a question about the samples you used. What kind of cells did you test? Were these treated to induce apoptosis? This antibody will only detect cleaved PARP, so if the cells are not apoptotic, the protein will not be detected (please see the second Western blot image, where the uninduced cells show no band, and the induced show a very nice band). The rest of the protocol looks fine to me. It may just be that the sample you are using is not supposed to have cleaved PARP. Please let me know if this helps and do not hesitate to contact us for further advice.
Answered on Sep 19 2006