Matrix metalloproteinases (MMPs) belong to a family of secreted or membrane-associated proteins capable of digesting extracellular matrix components. The importance of MMPs in tumor development and invasion as well as other diseases is well known.

AnaSpec is pleased to introduce the SensoLyte^TM 570 Generic MMP Assay Kit, offering the industry’s highest emission wavelength for MMP detection. The higher emission wavelength allows the SensoLyte^TM 570 Generic MMP Assay Kit to offer greater sensitivity in detecting enzyme activities with minimal interference from the autofluorescence of cell components and test compounds. This kit is ideal for detecting generic MMP activity in biological samples or for high throughput screening of MMP inducers and inhibitors using purified MMPs.

The SensoLyte™ 570 Generic MMP Assay Kit is optimized to detect the activity of a variety of MMPs, including MMP-1, 2, 7, 8, 9, 10, 13, and 14 (Fig.1). This kit uses a FRET peptide substrate labeled with 5-TAMRA (fluorophore) and QXL™ 570 (quencher) for continuous measurement of enzyme activities. In an intact FRET peptide, the fluorescence of 5-TAMRA is quenched by QXL™ 570. Upon the cleavage of the FRET peptide by MMPs, the fluorescence of 5-TAMRA is recovered, and can be continuously monitored at excitation/emission = 540 nm/575 nm. The assays are performed in a convenient 96-well format.

The Kit contains: • MMP 570 substrate • 5-TAMRA reference standard for calibration • APMA solution for pro-MMP activation • Assay buffer • Stop Solution.

Product	Size	Catalog #
SensoLyteTM 570 Generic MMP Assay Kit	1 kit	72101

Figure 1. Detection of MMP activities using the SensoLyte™ 570 generic MMP assay kit.

APMA-activated MMPs, 30 ng each, were individually mixed with 5-TAMRA/QXL™ 570 FRET peptide substrate and fluorescence was monitored after 1 hr. Endpoint signals were compared to background (FRET substrate without enzyme) to demonstrate MMP activities. The FRET peptide can detect the activity of nanogram range of all MMPs. (FlexStation 384II, Molecular Devices; Ex/Em=540nm/575 nm, cut off 570nm)

Table 1. Comparison of the newly introduced SensoLyte^TM 570 with the 520 Generic MMP assay kits

	SensoLyte™ 520 Generic MMP Assay Kit Cat# 71158	SensoLyte™ 570 Generic MMP Assay Kit Cat# 72101
FRET pair	5-FAM/QXL™ 520	5-TAMRA/QXL™ 570
Ex/Em (nm/nm)	490/520	540/575
Cleaved by	MMP-1, 2, 3, 7, 8, 9, 12, 13, and 14	MMP-1, 2, 7, 8, 9, 10, 13, and 14

Table 2. Publications citing the SensoLyte^TM 520 Generic Assay Kit.

This assay has been validated using purified MMPs; however, as the list below shows, MMPs from other sources have been used and reported by researchers.

Errami, M. et al. J. Pharma. Exp. Ther. DOI: 10.1124/jpet.107.133975 (2007).	General MMP activity (from rat heart) was measured using the SensoLyte™ (formerly Enzolyte) 520 Generic MMP assay kit.
Gueders, MM. Et al. Biochem. Pharmacol. 75, 514 (2008).	General MMP activity was assessed using the SensoLyteTM (formerly EnzoLyte) 520 Generic MMP Assay Kit…Proteins extracted from the crushed (mice) lung tissue were incubated with 1 mM APMA for 1 H in order to activate MMPs. 50 ul of sample were incubated in a 96 well plate at room temperature for 45 min.

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