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1. Order details: * Batch number: 140202 * Abcam order or Purchase order number: ab8639 * Antibody storage conditions (temperature/reconstitution etc) Working antibody store at 4?, Aliquot and store at -80? 2. Please describe the problem (high background, wrong band size, more bands, no band etc). Wrong band size. 3. On what material are you testing the antibody in WB? · Species: human, Huh7 cell transfected with pHBV-48. · Cell extract or Nuclear extract: cell extract · Purified protein or Recombinant protein: 3. The lysate * How much protein was loaded: 30 mg * What lysis buffer was used: RIPA lysis buffer (150 mM NaCl, 50 mM Tris pH 7.5, 10 mM EDTA, 1% NP-40, 0.1% SDS); Triple-detergent lysis buffer (150 mM NaCl, 50 mM Tris pH 8.0, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) * What protease inhibitors were used: Aprotinin, Leupeptin, PMSF * What loading buffer was used: 4X SDS sample buffer ( 250 mM Tris-HCl pH 6.8, 8% SDS, 20% b-mercaptoethanol, 40% Glycerol, 0.04% Bromophenol Blue) * Did you heat the samples: temperature and time: 99?, 10 min 4. Electrophoresis/Gel conditions/ Transfer conditions * Reducing or non reducing gel: SDS PAGE * Gel percentage : 12% * Transfer conditions: Buffer: 25 mM Tris, 192 mM Glycine, 20% Methanol 250 mA, 2 hr 5. Blocking conditions * Buffer: PBS * Blocking agent: milk, BSA, serum, what percentage: 5% skim milk * Incubation time: 1 hr * Incubation temperature: RT 6. Primary Antibody * Specification (in which species was it raised against): human hepatitis B virus core antigen · At what dilution(s) have you tested this antibody: 1:1000 or 1:500 · What dilution buffer was used: 5% skim milk/PBS · Incubation time: 1 overnight · Incubation temperature: 4? · What washing steps were done: TBS-T (0.5% Tween-20) wash 10 min for 3 times 7. Secondary Antibody * Specification (in which species was it raised against)? mouse * At what dilution(s) have you tested this antibody: 1:5000 * Incubation time 1hr * Wash steps: TBS-T (0.5% Tween-20) wash 10 min for 3 times * Do you know whether the problems you are experiencing come from the secondary? I don’t think so. 8. Detection method ECl, ECl+, other detection method: Immobilon TM Western Detection Reagents 9. Background bands · Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): no. · Is the blocking step sufficient? Yes. · Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) Yes · At what size are the bands migrating? Could they be degradation products of your target? 21 kDa. · Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) Fig.1. anti-HBc (Abcam, 10E11), 1:1000 Fig. 2. anti-HBc (Abcam, 10E11), 1:500 Fig. 3. anti-HBc (homemade rabbit polyclonal antibody), 1:1000 as a control. Protein samples on fig. 1 and fig.3 are the same. Fig. 4. anti-HBs ([another company] rabbit polyclonal antibody), 1:4000 as a control. Membrane in fig.1 was stripped and reprobe with anti-HBs antibody. Did you apply positive and negative controls along with the samples? Please specify. Positive control: HBV virion, negative control: Huh7 cell lysate 10. Optimization attempts · How many times have you tried the Western? 2 times · Do you obtain the same results every time e.g. are background bands always in the same place? Not exactly the same. · What steps have you altered? I have tried different concentrations (1:1000 and 1:500) and three kinds of protein extraction method: 1) RIPA-1: RIPA lysis buffer?scape the cells?Lysis on ice for 20 min?centrifuge 13,000 rpm, 20 min? Collect the supernatant and add sample buffer to the pellet and sonicate. 2) RIPA-2: RIPA lysis buffer?scape the cells?freeze and thaw?centrifuge 13,000 rpm, 20 min? Collect the supernatant and add sample buffer to the pellet and sonicate. 3) 3-dtgn: triple-detergent lysis buffer?scape the cells?freeze and thaw?sontcation?whole lysate.
Asked on Sep 01 2006
Thank you for providing extensive details of the customer's protocol, as well as images, it enabled me to assess very rapidly that there was a problem with the vial of ab8639 that you received. I can offer a replacement vial or credit note, whichever the customer prefers, thank you for letting me know what he/she would like,
Answered on Sep 04 2006