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Fluorometric Protease Assays in the SpectraMax Gemini Microplate Spectrofluorometer: Example Using Caspase3 (MaxLine Application Note #35)

background is also higher because white plates are inherently fluorescent.) Figure 5 gives an example of a kinetic caspase3 assay run in both a black and a white microplate. The slope of the reaction in the white plate was ~2.7 times that in the black plate. The initial fluorescence (background) was also ~2.5 times higher in the white plate. Despite the higher background, the white plate gave lower detection limits than did the black plate. Using the criterion of three positive SDs of the minus enzyme blanks, the estimated limits of detection for the assay were ~0.02 ng and 0.10 ng recombinant enzyme per well in the white and black plates respectively.


SUMMARY
Fluorogenic peptide substrates can interfere with the measurement of their own hydrolysis products. If the emission spectra overlap, the assay may have a high background. If the absorption spectra overlap, or if the absorbance of the substrate overlaps with the emission of the product, the substrate can quench the signal from the product. Therefore, both the excitation and emission wavelengths must be optimized to minimize interference from the substrate, while maintaining sufficiently high product fluorescence. SPECTRAmax GEMINI microplate spectrofluorometer facilitates the process by having dual monochromators that allow optimal excitation and emission wavelengths to be easily determined.


REFERENCES

1. Zimmerman, M., E. Yurevicz and G. Patel. 1976. A new fluorogenic substrate for chymotrypsin. Anal. Biochem. 70: 258262.

2. Gray, C.J. and J.M. Sullivan. 1989. Synthesis of 7amino4methylcoumarin (AMC) derivatives and their hydrolysis by plant cysteine proteinases. J. Chem. Tech. Biotechnol. 46: 1126.

3. Smith, R.E., E.R. Bissell, A.R. Mitchell and K.W. Pearson. 1980. Direct photometric or fluorometric assay of proteinases using substrates contai
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