Question (11953) | Rabbit Anti-Mouse IgG H&L (Alkaline Phosphatase) (ab6729)

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM loss of activity of the alkaline phosphatase SAMPLE amyloid fibers from a-beta: used anti-abeta antibody and than used the anti mouse IgG as secondary antibody PRIMARY ANTIBODY Abcam ab 10146 dilution 1:6000 SECONDARY ANTIBODY abcam ab 6729 dilution 1:1000 DETECTION METHOD UV-absorption POSITIVE AND NEGATIVE CONTROLS USED yes SAMPLE PREPARATION coated plates with amyloid, incubated with anti abeta and than with anti mouse IgG TRANSFER AND BLOCKING CONDITIONS BSA blocking HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 20 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes WHAT STEPS HAVE YOU ALTERED? incubation times and concentrations ADDITIONAL NOTES I tried the assay many times to find the right concentration, and eventually found it. the conentrations were in the described range for these antibodies. However suddenly the turnover rate of the alkaline phophatase came to almost a stand still within two weeks of using it. Normally is took 30 - 45 minutes to get the desired response. Now it takes 15-20 hours to get a comparable response, with the same time and concentrations used before. does the alkaline phosphase decompose after a certain period of time? I kept it at 4 degrees as was said in the data sheet.