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LOT NUMBER GR41994-1 ORDER NUMBER 39180 DESCRIPTION OF THE PROBLEM Multiple bands SAMPLE lanes 1,2, 3 and 4: human lung cancer cell line (Calu6)whole cell lysate, lanes 5 and 6: normal mammary epithelial cells (MCF10A)whole cell lysate, PRIMARY ANTIBODY anti-TIMP1 (ab61224) diluted 1/200 in PBS-Tween 0,1%, incubation ON at 4 celcius washed 4 times 5 min with PBS-Tw 0,1% DETECTION METHOD Enhanced Luminol (Perkin Elmer) film X-OMAT exposed 2 and 30 min POSITIVE AND NEGATIVE CONTROLS USED We consider MCF10A cells as positive control ANTIBODY STORAGE CONDITIONS aliquot the same day it arrived at the lab and stored at -20 SAMPLE PREPARATION lanes 1,2, 3 and 4 lysing buffer: HEPES 25mM pH7,5, NaCl 150mM, MgCl2 10mM, EDTA 1mM, Trition 0,1%, Glycerol 10%, aprotinin 10microM and sodium vanadate 1mM lanes 5 and 6 lysing buffer:sodium phosphate 20mM, NaCl 150mM, Triton 1%, EDTA 5mM, PMSF 0,2mg/ml, Aprotinin 10microg/ml, leupeptin 10microg/ml, sodium vanadate 0,25mg/ml Sample buffer 2X: Tris 150mM pH 6,8, SDS 1,2%, Glycerol 30%, beta-mercaptoethanol 15%, bromophenol blue 0,0018% Protocole: samples were mixed with sample buffer 2X (1:1) and boiled for 10 min AMOUNT OF PROTEIN LOADED lanes 1,3 and 5 70microg, lanes 2,4 and 6 20microg ELECTROPHORESIS/GEL CONDITIONS 12% resolving gel for denaturing SDS-PAGE and 4% stacking gel TRANSFER AND BLOCKING CONDITIONS wet transfer with Tris-Glycine-SDS buffer with 20% methanol, 900mA for 90min Blocking: PBS-Tween 0,1%- milk 5%, 90min, RT SECONDARY ANTIBODY anti-Rabbit-HRP (Santa Cruz sc-2004) 1/10000 in PBS-Tw 0,1% 90min washed 4 times 5 min with PBS-Tw 0,1% 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? No WHAT STEPS HAVE YOU ALTERED? We changed sample loading buffer. The one before contained less beta-mercaptoethanol and hight molecular weigth appeared on film ADDITIONAL NOTES Is it possible that we detect dimers of TIMP1
Asked on Nov 24 2011
Thank you for taking time to complete our questionnaire. I am sorry to hear that antibodies ab16123, ab39184 and ab61224 are not providing satisfactory results. The details provided will enable us to investigate these cases and will provide us with vital information for monitoring product quality. Having reviewed these 3 cases, I would like to offer some suggestions to help optimize the results from ab16123, ab39184 and ab61224 : To reduce the background, I recommend to load a maximum of 20µg of samples on the gel. I would suggest to use each primary antibody at a dilution 1/1000 and to dilute each one in the blocking solution. The dilution factor can then be optimized around this value. I would also recommend to try an incubation at room temperature for 2 or 3 hours. Some antibodies bind stonger and more specifically at room temperature. In order to determine the origin of the background I would recommend to run a no primary control in order to see if the background is due to some non-specific binding by the secondary antibody. Last but not least, when testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. Some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. An example of the above is the western blot obtained with the Abcam GAPDH antibody ab9385 : www.abcam.com/gapdh-antibody-hrp-loading-control-ab9385.html#GAPDH-Primary-antibodies-ab9385-3.jpg . Should the suggestions not improve the results, please do let me know. In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund. I hope this information is helpful, and I thank you for your cooperation.
Answered on Nov 24 2011