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Please see below the details of customer's complaint. Attached is the image of the gel. Antibody code: ab38307 Batch number: GR23921 Antibody storage conditions (temperature/reconstitution etc): -200C Description of the problem (high background, wrong band size, more bands, no band etc.): no bands Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.): Plasmodium falciparum - Cell extract. Sample preparation (Buffer/Protease inhibitors/Heating sample etc.): A fresh protein lysate was used each time, all procedure was performed on ice/40C, Parasites Cells were diluted in 96ul of ice cold PBSx1 and 4ul of protease inhibitor x25, after a 10min incubation 100ul of a 700C pre warmed sample buffer was added. Sample was place in 700C and after a few trails in 950C for 5min before loaded.. Amount of protein loaded: 10-20ul (concentration was not measured because the cell extract is not purified) Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.) Bio-Rad 4-20% gel (Mini-PROTEAN) Transfer and blocking conditions (Buffer/time period, Blocking agent etc.) Transfer: transfer buffer - 700ml DDW, 200ml methanol and 100ml transfer bufferx10 (Tris 478.8mM, Glycin 386.3mM, SDS 0.37%, Ph 8.3). 130 mA for 90min or 60min. Blocking: different blocking buffers were used: 5% skim milk or 5% BSA in 100ml PBS-T (0.1% tween or 0.04% tween) for 1h at room temp or overnight at 40C. Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Rabbit polyclonal to BSD (abcam ab38307), different ab dilutions were used: 1:2000 or 1:1000 in 5% skim milk 0.1% tween or in 1% skim milk and 0.04% tween. Incubation times: on shaker- 1h at room temp or over night at 40C. Different washes were preformed in PBS-T (0.1% tween): on shaker, 3 times x 10min or 3 times x 3min. Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Goat anti-rabbit IgG (H+L). different ab dilutions were used: 1:10000 in 5% skim milk 0.1% tween or in 1% skim milk and 0.04% tween. Incubation times: on shaker- 1h at room. Different washes were preformed in PBS-T (0.1% tween): on shaker, 3 times x 10min or 3 times x 3min. Detection method (ECL, ECLPlus etc.): Easy-ECL (Pierce). Exposure time at least 20min at room temp or overnight in 40C Positive and negative controls used (please specify): Positive control – a transgenic parasites line that is expressing BSD and is grown on 2ug/ml blasticidin. Negative control - a blasticidin non resistant parasite line. (Cannot grow on 2ug/ml blasticidin). Optimization attempts (problem solving) How many times have you tried the Western? 10 Have you run a "No Primary" control? No Do you obtain the same results every time? Yes, no bend except the ladder. What steps have you altered? Different loading volume of highly prodused sample, transfer time, blocking buffer and time, primary antibody dilutions and incubation times, wash times, secondary antibody dilutions and incubation times and different exposure times (as specified above).
Asked on Sep 21 2011
Thank you for contacting us. I am sorry to hear you have been experiencing problems with ab38307. The customer has made many changes to optimize and it is disappointing that the antibody is still not working. I would be happy to refund or replace the product. The only recommendation I would like to make is using 10% saponin for lysis, reviewing online protocols this is a common method to lyse Plasmodium falciparum and may be more effective than 1xPBS only. I hope this information helps. Looking forward to your reply on how to proceed.
Answered on Sep 21 2011