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For the technical support, Please, customer is having problems using human beta defensin antibodies and their respective recombinant proteins in WB applications. Find bellow the technical questionnaire filled out by the customer. As the beta defensins are small proteins (8 kDa), it seems this might be the origin of the problem. Maybe, the customer is losing these small bands during the running or even during the transfer. Could you please verify her protocol and give suggestions on how to solve this problem? Thank you in advance. Kind Regards, Abcam WB Questionnaire 1) Abcam product code ab14425 + ab50048 ab66072 + ab9872 ab19270 + ab50059 ab14419 + ab69499 2) Lot number ab14425: GR 17872-2 + ab50048: GR 45073-1 ab66072: GR 40785-1 + ab9872: GR 11654-6 ab19270: GR 730-1 + ab50059: GR 1609-2 ab14419: GR 21054-1 + ab69499: GR 3598-2 3) Antibody storage conditions (temperature/reconstitution etc) Antibodies: stored at -20 ºC. Recombinant proteins: after adding 100 ul PBS + 15% glycerol we aliquoted each one and stored at -80°C. 4) Description of the problem (high background, wrong band size, more bands, no band etc.) No bands using the beta defensin antibodies with their respective recombinant protein and no bands with lysate of chorioamniotic membrane. 5) Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.) Human recombinant proteins and lysate of chorioamniotic membrane tissue from human. 6) Sample preparation (Buffer/Protease inhibitors/Heating sample etc.) Lysis buffer: - 50mM Tris HCl pH 7,4 - 0,2mM NaCl - 0,1% Triton X-100 - 10mM CaCl2 - 10ul/mL Protease inhibitor from GE Healthcare Heating sample for 5 min at 100ºC. 7) Amount of protein loaded 30ug 8) Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.) 15% gel Running at 100 V for 1h30min Sample buffer (4x): - 250mM Tris-HCl 0,5M pH:6,8 - 8% SDS - 20% B-mercaptoethanol - 0,012g bromophenol blue -100 mL milli-Q water - 30% glycerol 9) Transfer and blocking conditions (Buffer/time period, Blocking agent etc.) Transfer: 1h30min at 120V Transfer buffer: 9,7g Tris, 57,6g glycine, 3,2L distilled water and 0,8L methanol Transfer membranes tested: 0,45µm Hybond membrane from Amersham and 0,22µm membrane from Millipore. Blocking conditions tested: 5% non-fat Milk diluted in TBS-T for 1h at room temperature under agitation. 5% non-fat Milk diluted in TBS-T for 2h at room temperature under agitation. 10% non-fat Milk diluted in TBS-T for 1h at room temperature under agitation. 10% non-fat Milk diluted in TBS-T for 2h at room temperature under agitation. 10) Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) 1. Human Beta Defensin-1: ab14425 (antibody) + ab50048 (rec protein) Tested dilutions: 1:500 and 1:250 for the antibody and 200 ng for the recombinant protein 2. Human Beta Defensin-2: ab66072 (antibody) + ab9872 (rec protein) Tested dilutions: 1:1000 and 1:500 for the antibody and 200 ng for the recombinant protein 3. Human Beta Defensin-3: ab19270 (antibody) + ab50059 (rec protein) Tested dilutions: 1:1000 and 1:500 for the antibody and 200 ng for the recombinant protein 3. Human Beta Defensin-4: ab14419 (antibody) + ab69499 (rec protein) Tested dilutions: 1:500 and 1:250 for the antibody and 200 ng for the recombinant protein All antibodies were diluted in TBST + 5% milk and incubated overnight at 4°C under agitation. We also tried to dilute them in TBST + 5% BSA. Wash step: 3x 5 min in TBST 11) Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Goat anti-mouse IgG HRP for ab14425, ab66072 and ab14419. Dilution: 1:5000 diluted in TBST +5% milk Incubation: 1 h Goat anti-rabbit IgG HRP F(ab’) fragment for ab19270. Dilution: 1:5000 diluted in TBST +5% milk Incubation: 1 h Wash step: 3x 5 min in TBST 12) Detection method (ECL, ECLPlus etc.) ECL Plus - Amersham 13) How many times have you tried the Western? 20 times 14) Do you obtain the same results every time? e.g. are the background bands always in the same place? The results we have are always the same for the 4 proteins. The respective bands of recombinant protein and sample do not appear. The running and transfer conditions are ok, because we have positive results with beta actin antibody in the same membranes. Also, the secondary antibody is working fine with beta actin. 15) What steps have you altered to try and optimize the use of this antibody? Primary antibody dilution, transfer membranes, blocking conditions...
Asked on Nov 30 2011
Thank you for contacting Abcam Technical Team and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is are having problems with 8 of our products. Special thanks for collecting the troubled customer's data/results in such an organized way. It may well be that the problem is either shipment/storage or protocol-related. Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem. 1) Arrival dates: Could you please confirm the exact dates when the customer received these items: - ab14425, ab50048, ab66072, ab9872, ab19270, ab50059, ab14419, ab69499 2) Orders and shipment: - Could you provide me the Abcam Order Numbers for these products? - Were these items shipped in the same package or not? 3) Marker bands: - What MW markers (i.e. range of the proteins for MW) were used for WB? I am particularly interested in the markers at the lower weight range. 4) Image: - Could you please attach an image representing the samples and the loading control on the same gels? 5) Secondary antibodies: - Has the customer used the respective secondary antibodies successfully with other primary antibodies? Could you please check if the problem does not come from the 2ndaries? 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 Nov 30 2011