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BATCH NUMBER 76164 ORDER NUMBER 61814 DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE Cell extracts PRIMARY ANTIBODY ab5986 was the primary antibody. Different dilutions were used form 1:500 to 1:5000. Antibody was diluted in TBS-Tween 5% milk. Incubation time: 1-1h30min Wash step: One rapid was and 3x10 min washes witn TBS-Tween. SECONDARY ANTIBODY Anti-rabbit IgG HRP (Amersham NA934V)was the secondary antibody. Different dilutions were used form 1:20000 to 1:50000. Antibody was diluted in TBS-Tween 5% milk. Incubation time: 1-1h30min Wash step: One rapid was and 3x10 min washes witn TBS-Tween. DETECTION METHOD Pierce Super Signal West Pico POSITIVE AND NEGATIVE CONTROLS USED Yes. Positive control: A431 lysate ab7909. Negative control: cell extract from bacteria. ANTIBODY STORAGE CONDITIONS Stored at +4?C SAMPLE PREPARATION Laemmli buffer has been used to prepare the samples. An equel volume of 2x Laemmli sample buffer was added. The sample buffer was complemented with b-mercaptoethanol. Samples are heated at 95?C for 5 min before loading them in the gel. AMOUNT OF PROTEIN LOADED 50-100 ng of target protein were loaded. ELECTROPHORESIS/GEL CONDITIONS 12% SDS-PAGE. TRANSFER AND BLOCKING CONDITIONS Tris-Glycine-methanol transfer buffer was used for running the gel. Gels were trasnfered for 45 min at 120V. The efficiency of trasnfer was determined by staining the membrane with Ponceau solution. Blocking reagent used was TBS-Tween buffer with 5% milk. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 15 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
Asked on Jan 18 2005
I'm sorry to hear you are having a problem with ab5986. I would like to suggest an overnight incubation of the antibody at 4C and maybe try a dilution of 1:300 as well. If you still have problems and your positive control (50ug of lysate per well) clearly shows that the antibody does not work please let me know and please send me an image of your blot and I will arrange for a replacement to be sent to you.
Answered on Jan 18 2005