SensoLyte® Viral & Bacterial Protease Assays
SensoLyte Viral Protease Assay Kits spotlight

AnaSpec is pleased to highlight a recently released bacterial protease assay kit, the SensoLyte® 520 Sortase A Activity Assay Kit, as well as previously released viral protease related assay kits, such as the SensoLyte® TEV (Tobacco Etch Virus), HCV (Hepatitis C Virus), HIV-1 (Human Immunodeficiency Virus-1) and WNV (West Nile Virus) protease activity assays.

SensoLyte® 520 Sortase A Activity Assay Kit

The SensoLyte® 520 Sortase A Activity Assay Kit is optimized for detecting Sortase A activity. This kit contains a novel internally quenched 5-FAM/QXL® FRET substrate. Sortase A cleaves the FRET substrate in two separate fragments resulting in release of 5-FAM fluorescence, which can be monitored at excitation /emission= 490/520 nm. The long wavelength fluorescence of 5-FAM is less interfered by the autofluorescence of components in biological samples and test compounds. The assays are performed in a convenient 96-well microplate format and can detect as low as 20 ng/mL active Sortase A.

Sortases are a family of membrane–anchored transpeptidases expressed by Gram–positive bacteria. Sortase A catalyzes the cleavage of C-terminal recognition motif (LPXTG) of multiple structurally unrelated proteins followed by formation of an amide bond with the peptidoglycan layer of the bacteria. Since Sortases are widely distributed among a variety of bacterial pathogens and required for virulence, they represent a promising therapeutic target for the development of novel anti-infective agents. In addition, Sortase A can be used as a biotechnology tool for a variety of protein modifications and immobilization by sortase-mediated protein ligation.

Sortase
Figure 1. Sensitivity of the SensoLyte 520 Sortase A Assay Kit was tested using serial dilutions of recombinant Sortase A. Assay can detect as low as 20 ng/mL of active Sortase A.

Product

Cat #

SensoLyte® 520 Sortase A Activity Assay Kit *Fluorimetric* NEW Ex/Em=490/520 nm

72228

Related Products: Sortase A protease, Recombinant NEW
Sortase Related Peptide Substrates

SensoLyte® 520 TEV Protease Assay Kits

The SensoLyte® TEV Protease Assay Kit uses a TEV specific substrate labeled with 5-FAM dye and QXL® 520 quencher. Proteolytic cleavage of this quenched 5-FAM-QXL® 520 conjugate yields a brightly green fluorescence, which can be continuously monitored at Ex/Em= 490/520 nm. Increase in fluorescence intensity is directly proportional to the TEV protease quantity/activity.

Sortase
Figure 2. Proteolytic cleavage of 5-FAM-QXL® 520 labeled TEV substrate. Fluorescence signal was measured with a fluorescence microplate reader (Flexstation 384II, Molecular Devices) at Ex/Em=490 nm/520 nm.

Product

Cat #

SensoLyte® 520 TEV Activity Assay Kit *Fluorimetric* NEW Ex/Em=490/520 nm

72227

SensoLyte® HCV Protease Assay Kits

Applying both peptide and fluorescent dye technologies, AnaSpec has developed an ultra-sensitive HCV NS3/4A protease FRET substrate that can detect the presence of HCV NS3/4A protease at levels of < 0.01 pmol. Based on an EDANS/DABCYL substrate, the donor and the quencher were substituted with 5-FAM and our trademark quencher, QXL® 520. This substrate, found in SensoLyte® 520 HCV Protease Assay Kit (Cat# 71145), shows a 22-fold lower Km compared to the EDANS/DABCYL peptide, the substrate found in SensoLyte® 490 HCV Protease Assay Kit (Cat# 71126 and 72087). These kits and the SensoLyte® 620 kit have been developed by AnaSpec for HTS screening of inhibitors and for measuring HCV NS3/4A activity.

Product

Cat #

SensoLyte® 520 HCV Protease Assay Kit *Fluorimetric* Ex/Em=490/520 nm

71145

SensoLyte® 620 HCV Protease Assay Kit *Fluorimetric* Ex/Em=590/620 nm

71146

SensoLyte® 490 HCV Protease Assay Kit *Fluorimetric* (200 assays) Ex/Em=340/490 nm

72087

SensoLyte® 490 HCV Protease Assay Kit *Fluorimetric* (500 assays) Ex/Em=340/490 nm

71126

Related Products: Recombinant HCV NS3/4A Proteases - wild-type and mutants wide selection
HCV Related Peptides

SensoLyte® HIV Protease Assay Kits

AnaSpec has developed three assay kits for measuring the activity of this protease and for HTS of its inhibitors. In each of the three SensoLyte® HIV assay kits, a substrate containing a different FRET pair is used. The general sequence of the peptide is derived from the native p17/p24 cleavage site on Prgag for HIV-1 protease. Incubation of recombinant HIV-1 protease with the substrate results in specific cleavage and a time-dependent increase in fluorescence intensity that is linearly-related to the extent of substrate hydrolysis. Due to its simplicity and precision in the determination of reaction rates required for kinetic analysis, these assays, offer many advantages over the commonly used HPLC or electrophoresis-based assays.

Product

Cat #

SensoLyte® 520 HIV Protease Assay Kit *Fluorimetric* Ex/Em=490/520 nm

71147

SensoLyte® 620 HIV Protease Assay Kit *Fluorimetric* Ex/Em=590/620 nm

71148

SensoLyte® 490 HIV Protease Assay Kit *Fluorimetric*
Ex/Em=340/490 nm

71127

Related Products: HIV-1 Protease, recombinant
HIV Protease FRET Substrate I

SensoLyte® 570 WNV Protease Assay Kits

The SensoLyte® 570 West Nile Virus (WNV) Protease Assay Kit provides a convenient, homogeneous assay for HTS of West Nile Virus NS3 protease inhibitors. It allows for continuous quantification of protease activity using a QXL® 570/5-TAMRA FRET peptide. In the intact FRET peptide, the fluorescence of 5-TAMRA is quenched by QXL® 570. Upon cleavage into two separate fragments by the WNV protease, the fluorescence of 5-TAMRA is recovered, and is monitored at Ex/Em = 540 nm/575 nm. The long wavelength fluorescence of 5-TAMRA shows less interference from autofluorescence of cellular components and test compounds. A lower emission wavelength assay, the SensoLyte® 440 WNV Protease Assay Kit (Ex/Em=354/442 nm) is also available.

Product

Cat #

SensoLyte® 570 West Nile Virus Protease Assay Kit *Fluorimetric* Ex/Em=540/575 nm

72080

SensoLyte® 440 West Nile Virus Protease Assay Kit *Fluorimetric* Ex/Em=354/442 nm

72079

Related Products: West Nile Virus NS3 Protease, recombinant

Custom Screening Service: AnaSpec provides custom screening services of inhibitors for proteases listed above, and many others. To request for a online quote, please click here. Click on the same link if custom assay development is needed.