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LOT NUMBER 937692 ORDER NUMBER 1026293 DESCRIPTION OF THE PROBLEM Non-specific band Actural western blot band size different from the predicted SAMPLE MCF-7, SK-BR-3, T47D, MDA-MA-231, MDA-MB-435 cell line PRIMARY ANTIBODY Concentration or dilution 1:300 Diluent buffer 2.5% BSA, PBST, Incubation time overnight Incubation temperature: 4°C DETECTION METHOD Supersignal west pico stable peroxide solution and luminal/enhancer solution(Pierce) were used according to manufacturer’s instructions. POSITIVE AND NEGATIVE CONTROLS USED no ANTIBODY STORAGE CONDITIONS -20C SAMPLE PREPARATION NUN buffer containing 0.33 M NaCl, 1.1 M urea, 1% Nonidet P-40, 25 mM HEPES Protease inhibitors: 1 mM DTT, 10 µg/ml leupeptin, 1 mM PMSF, 2 µg/ml aprotinin boling : yes AMOUNT OF PROTEIN LOADED 20 µg/lane ELECTROPHORESIS/GEL CONDITIONS 10% Tris Glycin gel TRANSFER AND BLOCKING CONDITIONS Type of membrane N.C membrane Protein transfer verified with Ponceau S solution Blocking agent and concentration 5% non-fat milk Blocking time 1hr Blocking temperature Room temperature SECONDARY ANTIBODY Pierce Donkey Anti-Rabbit IgG, (H+L), Peroxidase conjugated (product number: 31458) Isotype: IgG Concentration or dilution 1:7500 Diluent buffer 5% non-fat milk in PBST Incubation time 1hr Incubation temperature: Room temperature washing: PBST 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? Change the Primary antibody(Anti-GREB1 antibody) incubation condition from 1hr at RT to overnight at 4°C. ADDITIONAL NOTES I study about the mechanism of cholesterol specially LDLR(Low Density Lipoprotein Receptor) and PCSK9(Proprotein convertase subtilisin/kexin9). Recently, To verify the correlation between estrogen and PCSK9, I treated b-estradiol(sigma) in MCF-7, SK-BR-3, T47D, MDA-MA-231, MDA-MB-435 cell line respectively. Then I identify the protein expression of GREB1 as a marker of estrogen treatment. And GREB1 is very important molecule because it may be a regulator of PCSK9, I expect. So my research purpose is the regulation of GREB1 has an effect on the expression of PCSK9 and further LDLR, cholesterol level.
Asked on Feb 29 2012
Thank you for your enquiry regardingab72999 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer ishaving problems with this antibody.
Could you please specify what size are the non-specific bands? Are they lower or higher than the expected size (216 kDa)?
Could you attach an image (saved as a jpeg file) to your e-mail so that I can have a look at the bands.
Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.
Answered on Feb 29 2012