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BATCH NUMBER 77713 ORDER NUMBER ? DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE whole blood looking at cytotoxic cells: CD3+CD8+, CD3+CD8+CD56+ and CD3-CD56+ cells PRIMARY ANTIBODY ab15723 goat ab and used goat IgG for isotypic control. used 15 minute RT incubation times. followed Intraprep procedure SECONDARY ANTIBODY ab7004 donkey anti goat - PE DETECTION METHOD FACScalibur POSITIVE AND NEGATIVE CONTROLS USED cytotoxic cells from whole blood should be positive. B cells from whole blood should be negative ANTIBODY STORAGE CONDITIONS 4 degrees SAMPLE PREPARATION none NUMBER OF CELLS USED 100ul whole blood PERMEABILIZATION STEP used Beckman Coulter permeabilization product Inraprep BLOCKING CONDITIONS none HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? added more primary antibody, added more secondary antibody ADDITIONAL NOTES Have you run this ab at all using flow cytometry? ANY help would be appreciated. Different permeabilization method? Longer incubation times?
Asked on Jan 25 2005
Thank you for your enquiry regarding ab15723. As stated on the datasheet, ab15723 is a Fast-Track antibody. Fast-Track antibodies are immunogen affinity purified and are shown by ELISA to have high titre values against their immunogen. We are currently testing them for use in Western blotting. At this stage, there can be delays. We sometimes experience problems in searching for / developing the correct lysate or knock-out cell-line. In addition, certain products can be difficult to characterise, due, for instance, to low expression levels or a lack of expression in easily available lysates. We realise that these products might be very suitable for your research and do not wish to delay this; therefore, we release them early with the following understanding: As Fast-Track antibodies are not yet fully characterised we are unable to offer our normal level of technical support. We cannot guarantee that the antibody will work in any application other than ELISA against the immunizing peptide and therefore we are unable to offer a refund if the antibody does not work in your application. Ab15723 has not been tested yet in FACS; there is only some preliminary results at this point for Western blotting. I'm afraid that I can only suggest at this point to increase the amount of primary antibody that you are using, and increase the incubation period. You should know that we would greatly appreciate your feedback on these antibodies, whether positive or negative. We will award $300 of Conference Travel Money to the first researcher who sends us positive feedback (including an image and details of materials & methods). Conclusive negative feedback that leads us to withdraw the antibody will also be rewarded with $300 of Conference Travel Money. If you have any additional questions, please contact us again.
Answered on Jan 26 2005