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BATCH NUMBER 150694 ORDER NUMBER DBI/253 DESCRIPTION OF THE PROBLEM No signal SAMPLE HeLa cells total extracts PRIMARY ANTIBODY we tried dilution form 1:100 to 1:1000 in PBS and we incubated ON in 5% defatted Milk at 4?C DETECTION METHOD ECL Plus POSITIVE AND NEGATIVE CONTROLS USED Positive control. we used another primary antibody that was working pefectly with this protocol and with the secondary antibody that we used ANTIBODY STORAGE CONDITIONS 4?C SAMPLE PREPARATION we addd to the cell pellet Laemly buffer, heat 5 min at 95?C and load on a SDS-PAGE AMOUNT OF PROTEIN LOADED The extracts ehould br round 25 micrograms/lane ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE, 12% acrylamide TRANSFER AND BLOCKING CONDITIONS semidry transfer checked with Ponceau Blocking with 5% Milk for 1 hr at room temperature SECONDARY ANTIBODY [another company], anti rabbit HRP; 1:5000 dilution in PBS, 1 hr incubation time at room temperature HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? We have tried different dilution of the antibody and we also used more cells for a more reach extract without success.
Asked on Mar 17 2006
Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. You have followed the protocol recommended by our datasheet including the dilutions and blocking agent. Furthermore Eap30 expression has been shown in HeLa cells in the following publication: Martin-Serrano J, Yarovoy A, Perez-Caballero D, Bieniasz PD. Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins. Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12414-9. Epub 2003 Sep 30. PMID: 14519844 However, my concern is that the detection of Eap30 has to my record only been shown at the transcript level by PCR amplification and therefore the level of protein expression may be insufficient for western immunoblot detection. I would therefore like to recommend that you incorporate a positive control in your experiments. I would like to recommend that the human hepatoma cell line HepG2 are used as a positive control; as shown in the western blotting image on our datasheet. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.
Answered on Mar 21 2006