Because DPPIV/CD26 protein levels may not accurately reflect the levels of active DPPIV enzyme, it is useful to measure DPPIV activity rather than performing DPPIV immunoassay. The DPPIV/CD26 Assay kit for biological samples is a complete assay system designed to measure DPPIV activity in biological fluids such as plasma, serum, urine, and saliva. Uses for the kit include correlation of DPPIV activity with disease states or determination of the efficacy of DPPIV inhibitors administered in vivo. The kit can easily be used for other biological fluids such as tissue, live cells and cell extracts, and exudates. Enough reagents are provided to perform at least 96 assays. The kit contains both a chromogenic substrate (H-Gly-Pro-pNA) and a fluorogenic substrate (H-Gly-Pro-AMC). Cleavage of the p-nitroaniline (pNA) from the chromogenic substrate increases absorbance at 405 nm. The fluorimetric assay is based on the cleavage of 7-amino-4-methylcoumarin (AMC) moiety from the C-terminus of the peptide substrate, which increases its fluorescence intensity at 460 nm.
Colorimetric detection, Fluorescence microscopy. Correlation of DPPIV activity with disease states, or determination of the efficacy of DPPIV inhibitors administered in vivo.
DPPIV enzyme (Human, recombinant) (5 mU; One U=1μmole/min at 37°C, 100µM H-Gly-Pro-pNA. Purity >95% (SDS-Page)), Storage: -70°C; avoid freeze thaw cyclespNA substrate (H-Gly-Pro-pNA) (55µl; 100mM in DMSO)Storage: -70°CpNA calibration standard (p-nitroaniline) (600μL; 80µM in 50mM Glycine, pH 8.7, 1mM EDTA)Storage: -70°CAMC substrate (H-Gly-Pro-AMC) (45µl; 50mM in DMSO), Storage: -70°CAMC calibration standard (7-amino-4-methylcoumarin) (600μL; 50µM in 50mM Glycine, pH 8.7, 1mM EDTA), Storage: -70°CInhibitor P32/98 (Prod. No. BML-PI142-9090) (20µl; 1mM in DMSO), Storage: -70°CAssay buffer (20ml; 50mM Glycine, pH 8.7, 1mM EDTA; liquid in screw-cap plastic bottle), Storage: -20 or -70°C½-volume clear microplate, Storage: Room temperature½-volume NBS white microplate, Storage: Room temperature