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I am not seeing any band in human endothelial cells. I load 30 ug, tried denatured, reducing conditions, incubated primary overnight at 4 degrees, tried 1/500-1/1000. Do I need to use non-reduced, non-denaturing conditions? Can you send me a picture of the blot you did?
Asked on Aug 22 2005
Thank you for contacting us for technical support regarding ab759. I have contacted the laboratory and was able to find out that indeed non reducing conditions are advised with this antibody. I have added immediately on our datasheet (accessible below) this information and the image of the blot where four lysates were tested and where you can see a clear band of beta 2 Microglobulin with two lysates (U-937 cell lysate and Raji cell lysate). We expected a signal on Jeg3 cells since on the native lysate it worked by ELISA (ab759 as detecting antibody, directly conjugated with HRP). However, for WB condition, the lysate was not reduced (only denatured by SDS), which may explain the lack of signal with this lysate (furthermore each lysate preparation was not done at the same time). Therefore collectively, the antibody works in western blotting in non reduced conditions and you may find you can achieve a signal with non reducing conditions. May I also suggest to use a 15 or 20% gel to achieve a good separation and run a positive control of U-937 cell lysate or Raji cell lysate? We used a concentration of 2ug/ml of ab729, incubating overnight and blocking for 2hrs with 5% non fat milk. I hope this information will help you, please do not hesitate to contact me again if I can be of further assistance,
Answered on Aug 24 2005