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DESCRIPTION OF THE PROBLEM High background, multiple bands and wrong band size SAMPLE mouse stomach sonicated in RIPA buffer plus complete protease inhibitors (Roche # 1697498) on ice. The whole tissue extract was separated on 15% SDS-PAGE and thansfered to PVDF membrane. PRIMARY ANTIBODY Ab1257 goat anti-ddddk 1:1000, 1: 2000, 1: 5000, 1:10000 and 1: 15000 in PBS-T with/without 5% non-fat milk incubated for 1 hr/RT wash in PBS-T 10 minx3 time SECONDARY ANTIBODY Sigma rabbit anti-goat, HRP conjugated 1:2000, 1: 5000 in PBS-T incubated for 30 min to 1 hr/RT wash in PBS-T 10 minx3 times DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED Negative control: stomach extract without my transgene, HeLa cell extract Positive control: HeLa cell with my transgene ANTIBODY STORAGE CONDITIONS 4 C cold room in original package SAMPLE PREPARATION RIPA buffer plus complete protease inhibitors (Roche # 1697498). AMOUNT OF PROTEIN LOADED 20 ug/well ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE, reducing, 15% precast gel from BioRad TRANSFER AND BLOCKING CONDITIONS Stander semi-dry transfer buffer Blocking in 5% non-fat milk HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? >10 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? primary Ab dilution, secondary Ab dilution ADDITIONAL NOTES I used Sigma M2 Ab (monoclonal from mouse) as golden standard for FLAG tag detection. Now I turned to your product because it's cheaper. In the whole series of my western, The M2 Ab gives me a sharp, solid single band without background, which means there is no degradation in my sample.
Asked on Feb 12 2004
Results such as these are commonly solved by loading less lysate (try 5 mcg/lane), diluting the primary antibody more (try 1:25,000) and diluting the secondary antibody more (try 1:20,000). The goat anti-ECS is 10 to 20 fold higher titered than the Sigma M2 antibody. You could use the Sigma M2 antibody at 1:25 and would likely get results similar to those you are seeing with the goat anti-ECS.
Answered on Feb 23 2004