In the Fluorescent Protease Assay Kit (Cat. No. 23266), I had poor reproducibility within my plate or across experiments. What could be the cause?
Variations in pipetting can directly contribute to assay error, especially in the FRET-mode, because the working reagent itself has some intrinsic background fluorescence. Use reverse-pipetting or other techniques to prevent the introduction of small air bubbles into the plate wells. In FP-mode, if the sample matrix contains very high amounts of interfering fluorescent material, better quality results may be obtained by subtraction of buffer blanks from the data used for the mP calculation. In addition, FP detection is very sensitive to the position of the optical head in relation to the center of the well with respect to its X and Y coordinates, especially with 384-well plates. This position-artifact may be identified as reproducible patterned data by positioning the plate for a second reading 180 degrees relative to the first reading. Ensure that the instrument read positioning settings are appropriate for the type of plate chosen.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.