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BATCH NUMBER 111955 ORDER NUMBER 103/8 DESCRIPTION OF THE PROBLEM No band which correspond to synphilin-1 SAMPLE HEK 293 cells transfected with WT-synphilin-1 (V5-tagged) or empty vector PRIMARY ANTIBODY Dilution at 1/1000 in 5% non-fat milk Incubation over night Wash with PBS-Tween DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED Positif control: Cells transfected with WT-synphilin-1 V5 tagged and detect with anti-V5 wath indicates transfection is OK Negatif control: Cell transfected with empty vector ANTIBODY STORAGE CONDITIONS Aliquots 10?l at 70?C SAMPLE PREPARATION Lyse buffer: Tris 20mM with proteases inhibitors cocktail AMOUNT OF PROTEIN LOADED 80?g ELECTROPHORESIS/GEL CONDITIONS Gel 10% TRANSFER AND BLOCKING CONDITIONS Transfers during 2hours Blocking with 5% non-fat milk during 30min SECONDARY ANTIBODY Anti-rabbit at 1/5000 in 5% non-fat milk Incubation during 4hours 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 |
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ANSWER: |
Thank you for your enquiry and for filling out our technical questionnaire so comprehensively. I am sorry to hear that you have been having difficulties with this antibody. Your data is very conclusive and seems to suggest that this antibody cross reacts against something other than your V5-synphilin construct. Quality is important to Abcam. We operate a 90 day replacement or refund policy from the date of delivery should a product not behave as on the datasheet. If your purchase meets these criteria, please can you e-mail me the details of your order including the date or purchase and PO number. I look forward to hearing from you. |
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Does this antibody detect Synphilin in Western blotting in whole brain homogenates and how many bands are there? Does it recognize mouse Synphilin? |
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ANSWER: |
Synphilin-1 cannot be detected in whole brain homogenates without immunoprecipitation first. When the antibody is used to immunoprecipitate material from whole rat brain, the major band is at approximately 90kDa and faint bands at approximately 60 and 40kDa. Mouse tissue has never been tested by us. |
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