Cut-N-Glow™ HIV Protease Activity Kits

$395.00

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Capase 3

Application

This kit is specific for the HIV Protease, a retroviral aspartyl protease that is critical to viral life-cycle. The Cut-N-Glow approach involves the introduction of a structural distortion into one of the complementary fragments (GFP 11), through the use of a conditionally stable tether, which serves to constrain the N and C termini of GFP 11 closely in space, thereby, diminishing the mutual affinity of the two fragments and blocking protein self-assembly until the tether is cleaved. The distortion can be reversed upon site-specific proteolysis of the tether, resulting in GFP 11 fragment assembly with GFP 1-10, generating reconstituted, functional GFP. Depending on the tether, the chimeric GFP can serve as a substrate for proteases from the three major classes: serine, cysteine, and aspartic acid.

Suitability

This kit contains sufficient reagents for one 96 well plate (96 tests)


 


Background:

Proteases occur naturally in all organisms and are valuable tools in medical diagnostics serving as initiators of cell signaling, as regulators of immune responses, and as agents of infectious disease. Therefore, mapping proteases in parasitic diseases and bacteria as well as assayable proteases associated with cancer could lead to the identification of shared structural similarities validating potential drug targets. This strategy utilizes split proteins in a conditionally inactive form with the aid of a conformational distortion maintained by a cleavable tether. This method is applied to convert split GFP into a latent fluorophore that can be activated by site-specific proteolysis. The chimeric GFP serves as a substrate for representative enzymes from the three major protease classes: serine, cysteine, and aspartic acid.

This kit is specific for HIV Protease, a retroviral aspartyl protease that is critical to viral life-cycle, and contains sufficient reagents for one 96-well plate (96 tests).

Assay

The Cut-N-Glow™ approach involves the introduction of a structural distortion into one of the complementary fragments (GFP 11), through the use of a conditionally stable tether, which serves to constrain the N and C termini of GFP11 closely in space, thereby diminishing the mutual affinity of the two fragments and blocking protein self-assembly until the tether is cleaved. The distortion can be reversed upon proteolysis of the tether, resulting in fragment assembly with GFP 1-10, generating reconstituted, functional GFP.



REAGENTS

Components Supplied: (Sufficient reagents have been supplied for 96 individual tests)
Constrained substrate, 1.0 mL. Supplied ready to use at approximately 225 µg/mL.
Store -20C.  
Detector (S1-10): Complementary GFP fragment. 20 mL. Supplied ready to use at approximately 1.0 mg/mL.. Store -20C.
Positive Control Reagent, 500 µL. Supplied ready to use. Store -20C.

Note: When stored at -20C, the reagents are stable until the date indicated either on the box or on each component.  Depending on the particular usage requirements, it may be appropriate to re-aliquot reagents to smaller working volumes to avoid repeated freeze-thawing or repeated pipetting from the same vial.

Materials required, but not supplied:
TE buffer pH 2.0
HIV protease: recommended Sigma cat# SRP2152 as an internal control.
96 well microplate compatible with customer's UV plate reader.
UV plate reader
Humidified incubation system


In Vitro ASSAY

1) Equilibrate kit components to ambient temperature.
2) In a microplate well, mix constrained substrate 1:1 (10 µL:10 µL) with TE buffer, pH 2.0. Repeat for      the number of wells as needed.
3) Add 0.5 µL of HIV protease to reagent control well.
4) Add 1.0-10 µL of test sample to remaining wells.
5) Incubate overnight at 37⁰C in a humidified incubator.
6) Add 200 µL of detection reagent to test and controls wells and incubate plate at 37⁰C for 6-16 hrs.
7) Measure fluorescence at 535 (+ 25) nm using 485 (+25) nm excitation.

DATA ANALYSIS
Data Analysis
Subtract the blank fluorescence values from the final fluorescence values of the sample(s) and the positive control. Perform appropriate statistical analysis, if applicable.


REFERENCES
1. Protease Activation of Split Green Fluorescent Protein. Callahan, B.P., Stanger, M.J., Belfort, M. ChemBioChem ‐ 2010 Nov 2;11(16):2259-63

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