Cut-N-Glow™ is the first fully biological in vivo protease mapping tool that emits fluorescence. This assay is easily tailored via standard cloning techniques to detect specific proteases or protease inhibitors or to map protease substrate preference in vitro or in vivo. Chemical synthesis is not required and there is no need for co-factors or co-substrates. Additionally, this assay only requires two reagents and both are proteins that can be easily obtained following over-expression of E. coli.
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.
CASP-3 is a member of the Caspase family (cysteine-aspartic acid protease) and when activated, is an essential factor in cell apoptosis.
This kit is specific for Capsase-3 and contains sufficient reagents for one 96-well plate (96 tests).
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.
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.
Detector (S1-10): Complementary GFP fragment. 20 mL. Supplied ready to use at approximately 1.0 mg/mL.. Store -20C.
Positive Control Reagent, 500 µL. Supplied ready to use. Store -20C.
Note: When stored at -20C, 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:
Caspase3 , Human: recommended Sigma cat# C1224 as an internal control
96 well microplate compatible with customer's UV plate reader.
UV plate reader
Humidified incubation system
In Vitro Complementation assay
1. Equilibrate kit components to ambient temperature.
2. In a microplate well, mix 10 µL constrained substrate with 200 µL of detection reagent, and allow to equilibrate at ambient temperature for 2 h. Repeat for the number of wells as needed.
3. Add 0.5 µL of Caspase-3 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, if necessary.
6. Measure fluorescence at 535 (+ 25) nm using 485 (+25) nm excitation.
Subtract the blank fluorescence values from the final fluorescence values of the sample(s) and the positive control. Perform appropriate statistical analysis, if applicable.
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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