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BATCH NUMBER 176737 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE I have transfected the DNAs encoding either APP or factor A into human cancer cells. The number of cell was counted and resuspended in appropriate amount of 2X SDS-PAGE sample buffer and boiled for 3min. PRIMARY ANTIBODY The membrane was blocked with 5% skim milk in TBS-T buffer. Anti-APP antibody was diluted at first 1:500 and later 1:100 and added to the membrane and incubated overnight with gentle rocking. The membrane was incubated further 30min at RT, washed three times with TBS-T DETECTION METHOD After 3 times washing (10min for each washing), ECL solution (Amersham-Pharmacia) was added, incubated for 1 min and the membrane was developed with Hyperfilm (Amersham-Pharmacia) by exposing the film for 5 min, 20 min, 2 hours, or overnight POSITIVE AND NEGATIVE CONTROLS USED I also tried to detect endogenous APP in the same cell line by using another company's antibody and there was no problem for detection. ANTIBODY STORAGE CONDITIONS 4C ELECTROPHORESIS/GEL CONDITIONS 8% SDS-PAGE TRANSFER AND BLOCKING CONDITIONS transferred activated PVDF membrane for 90 min at 60V SECONDARY ANTIBODY secondary antibody (anti-mouse) was added and incubated for 2 hours. 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? Yes WHAT STEPS HAVE YOU ALTERED? See above ADDITIONAL NOTES While i was filling the complaint report, i repeated the experiment with fresh primary and secondary antibody and i still could not get the target band. There were several faint background bands but no APP specific band.
Asked on May 08 2006
Thank you for your enquiry. I am sorry to hear that your customer has been having difficulties using this antibody by western blotting. Their approach is largely on that I would recommend. They have tried various dilutions using an overnight incubation. I am certainly prepared to offer your customer credit if the antibody was purchased within the past 90 days. If this is the case please e-mail me details of the order including the date of purchase and order number.
Answered on May 10 2006