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BATCH NUMBER 210603 ORDER NUMBER 250420 DESCRIPTION OF THE PROBLEM No signal: I have used Ab21236 at 1:000 and 1:500 dilution in 0.1%TTBS in a Western blot of HEK 293 cells expressing epac1 with cAMP sensors. I did not obtain any signals other than magic marker molecular weight proteins indicating 2 nd antibody reactivity. I tested the antibody 8/30/07 and again yesterday to confirm results obtained using an anti-epac1 ab from another source. I checked to see if any reports were sent regarding this antibody. No reports were submitted by other investigators. What has been your experience with your antibody. What positive controls are you using? i.e. kidney, cardiac or brain? We are testing your antibody not only against expressed epac1 but looking at mouse cardiac, brain and parotid tissue fractions. SAMPLE HEK-293 recombinant epac1 with cAMP sensors/mouse whole cell lysates, heart, brain, parotid PRIMARY ANTIBODY Ab21236 Abcam/rabbit/0.1%TTBS/1:500, 1:1000/ 1 and 2 hours for primary;1 h for 2ndary. Standard wash protocols using with 0.1 TTBS DETECTION METHOD Amersham ECL/Pierce ECL POSITIVE AND NEGATIVE CONTROLS USED Positive: HEK 293 cells expressing epac1 with cAMP sensors. ANTIBODY STORAGE CONDITIONS -20 C, aliquoted for Ab21236 and -80 C, aliquoted for 2ndary SAMPLE PREPARATION NuPAGE sample buffer at 1x/multiple, group specific protease inhibitors/65 C for 15 min. AMOUNT OF PROTEIN LOADED Sufficient to give strong signals using 3 other antibodies from different sources but with only 77% (or less) homology to mouse epac1. We wanted to test your antibody because your epitope antibody corresponds to 15/16 aa of mouse sequence and more than one immunoreactive peptide was observed between 150 and 100 Kd . Other tissue: mouse whole cell lysates up to 100 micrograms protein gel load. ELECTROPHORESIS/GEL CONDITIONS Tris-Glycine SDS/6%PAGE; reducing TRANSFER AND BLOCKING CONDITIONS 1/2 Towbain/30 volt constant/overnight at 4 C/1%BSA,1%PEG,1%PVP in 0.1%TTBS, 1 h at rt SECONDARY ANTIBODY Jackson ImmunoResearc/Donkey/1:1000, 1:20000/1 hour/ standard wash protocols 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? primary ab dilution
Asked on May 21 2007
I'm very sorry for the delay in replying to you. I have now received feedback from the laboratory that tested the antibody ab21236 and was informed that it was tested against recombinant Epac 1 and gave a very good strong band. However, it had poor affinity for epac 1 protein when it was tagged with a marker protein (Myc-tag). Similar results were obtained with His-tag. We believe this also depend upon the orientation of the tag marker. We are not aware of a suitable positive control of endogenous Epac1 but can provide in our catalogue the recombinant protein used to test this antibody. Please let me know if you would be interested and I will add this to the catalogue. We would recommend to try to immunoprecipitate the cell extract form 250ug-500ug total protein to enrich the sample and then do a western as I have been informed that this antibody does immunoprecipitate well. Furthermore, we recommend to incubate the primary antibody overnight at 4C and to boil the samples at 95C to make sure they are well denatured. Please make sure you are using reducing conditions in the loading buffer and that your protease inhibitors are fresh. Finally, the blocking buffer sometimes can alter the signal, it would therefore be good to try a different blocking buffer, milk or BSA (typically we use 5%BSA for 1 hour and incubate the antibodies in TBST only). I hope this information will help you, please do not hesitate to contact me if I can be of further assistance,
Answered on May 21 2007