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BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM target band shape is not good and more bands and detected markers. SAMPLE U937 cell extract PRIMARY ANTIBODY abcam/rabbit/5% blocking solution/1:1000/ overnight/ TBST wash 15min 5 times DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED Use positive control SNU16 and U937 // negative 293T ANTIBODY STORAGE CONDITIONS -20 degree SAMPLE PREPARATION Sigma M lysis buffer / roche protease inhibitor cocktail/ heating 100 degree 5min AMOUNT OF PROTEIN LOADED First 20ug and antibody detected many bands so diluted sample 1/10, 1/50/ 1/200 ELECTROPHORESIS/GEL CONDITIONS 10% gel, reducing gel TRANSFER AND BLOCKING CONDITIONS Semi dry transfer 40min/ 5% skim milk blocking SECONDARY ANTIBODY santacruz/5% blocking solution/1:10000/ 1hour RT HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Antibody dilution and sample dilution and blocking solution percentage ADDITIONAL NOTES I want to know the concentration of this antibody, ug/ul.
Asked on Jan 15 2007
Thank you for taking the time to submit these comments. It looks to me from the gel that the gel has not run out properly. Could you please enquire with the customer as to how the gel was made and how long the running gel and stacking gel were left to polymerise for prior to use? Smeared banding such as that shown can be due to running the gel at too high a voltage. Since the gel can loose rigidity creating band smearing. Under what conditions was the gel ran? I suspect that reducing the voltage or running the gel in the cold room would reduce the observed smearing. I look forward to hearing from you again shortly.
Answered on Jan 15 2007