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BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 210605 DESCRIPTION OF THE PROBLEM No specific signal, often nothing at all. SAMPLE Human T cells as well as PBMC depleted of monocytes and NK cells PRIMARY ANTIBODY ab11905, abcam, 1:1000. Incubation from 2 hr RT to overnight 4 degrees. 2 hr wash before addition of secondary. DETECTION METHOD ECL plus POSITIVE AND NEGATIVE CONTROLS USED Positive control: Jurkat lysate. No negative control ANTIBODY STORAGE CONDITIONS storage -20 degrees, aliquoted in two lots of 25 ?l SAMPLE PREPARATION lysed samples (buffer: 9 ml 5% triton x-100 in Tris HCL, 9g/l NaCl + 1 ml 0.5 M EDTA + complete protease inhibitor tablet (ROCHE)) mixed 1:1 with l?mmli buffer. AMOUNT OF PROTEIN LOADED Varied, sometimes different amounts of same sample used on 1 blot. ELECTROPHORESIS/GEL CONDITIONS 4-12% Nupage gel (Invitrogen), MOPS or MES buffer TRANSFER AND BLOCKING CONDITIONS Transfer 3 hrs, 4 degrees in transfer buffer (Invitrogen), 10% Methanol., blocked with 1 % BSA, 1 hr. SECONDARY ANTIBODY anti-rabbit-HRP, amaxa. 1:7000, 2 hr. wash two hr before development. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Incubation time and temperature, amount of sample, type of cell lysate, development time ADDITIONAL NOTES About purchase order number: The antibody was ordered by our technician, together with anti-RUNX1 (PON zumkehr210605. I have used the anti-RUNX1 antibody (ab11903) to satisfaction on the same samples and even blots as those used for this anti-RUNX3 antibody, also at a dilution of 1:1000 and with the same reagents. The much smaller amount of anti-RUNX3 supplied did not allow for dilutions less than 1:1000, without skyrocketing costs.
Asked on Jul 21 2005
Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with ab11905. For Western blotting, we do suggest using this antibody at 1:500 - 1:1000 and so I do suggest trying ab11905 at 1:500 with incubation overnight at 4C. We also suggest using Raji nuclear extract as a positive control. I hope this helps. If you need additional assistance, then please contact us again.
Answered on Jul 25 2005