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DESCRIPTION OF THE PROBLEM Multiple bands SAMPLE •Species: human •What’s cell line or tissue: H1299 and HCC827 •Cell extract or Nuclear extract: cell extract PRIMARY ANTIBODY •Species: rabbit •Reacts against: human •At what dilution(s) have you tested this antibody: 1ug/mL •What dilution buffer was used: 3% BSA •Incubation time: O/N •Incubation temperature: 4℃ •What washing steps were done: 5min x 3 repeats, 10min x 2 repeats DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED Yes, I stripped the membrane, and then hybridized with antibody against v5 tag. ( I transfected v5-taged plasmid to the cells) ANTIBODY STORAGE CONDITIONS -20℃ SAMPLE PREPARATION •What lysis buffer was used: M-Per •What protease inhibitors were used: no •What loading buffer was used: 5x sample buffer •Phosphatase inhibitors : no •Did you heat the samples: temperature and time: 100℃ , 5min AMOUNT OF PROTEIN LOADED 50ug ELECTROPHORESIS/GEL CONDITIONS •Reducing or non reducing gel: non-reducing •Reducing agent: •Gel percentage : 10% TRANSFER AND BLOCKING CONDITIONS Transfer conditions: (Type of membrane, Protein transfer verified): PVDF, 400mA, 1hr10min •Buffer: 3% BSA •Blocking agent: milk, BSA, serum, what percentage: 3% BSA •Incubation time: 1hr •Incubation temperature: RT SECONDARY ANTIBODY •Species: goat •Reacts against: rabbit •At what dilution(s) have you tested this antibody: 1/5000 •Incubation time: 1hr •Wash steps: 5min x 3 repeats, 10min x 2 repeats •Fluorochrome or enzyme conjugate: HRP •Do you know whether the problems you are experiencing come from the secondary? no HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? no
Asked on Sep 30 2011
Thank you for contacting us. I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. Having reviewed your protocol and I have a few suggestions that may improve the results that you are currently seeing. Firstly, can I ask if there is a reason for not using a reducing agent? Adding DTT or b-mercaptoethanol can help to denature the protein by reducing the disulphide bonds in the protein. Additionally, I would strongly recommend using a protease inhibitor with the lysis buffer used as the M-Per lysis buffer does not appear to contain any itself. This should significantly reduce any degradation of the protein seen. Often altering the blocking agent used can also change the non-specific bands seen, in this case using 5% milk instead of 3% BSA may prove beneficial. I hope this information is helpful to you. Please do not hesitate to contact us if you continue to experience problems with this antibody, I will be happy to arrange a replacement of credit note for this product.
Answered on Sep 30 2011