Amplite® Universal Fluorimetric Protease Activity Assay Kit Green Fluorescence

Monitoring of various protease activities has become a routine task for many biological laboratories. Our Amplite® Universal Fluorimetric Protease Activity Assay Kits are an ideal choice for performing routine assays necessary during the isolation of proteases, or for identifying the presence of contaminating proteases in protein samples. The kits use fluorescent casein conjugates that are proven to be a generic substrate for a broad spectrum of proteases. In the intact substrate, casein is heavily labeled with a fluorescent dye, resulting in significant fluorescence quenching. Protease-catalyzed hydrolysis relieves its quenching effect, yielding brightly fluorescent dye-labeled short peptides. The increase in fluorescence intensity is directly proportional to protease activity. The kits provide all the essential components with an optimized "mix & read" protocol that can be easily automated to HTS instruments.

Example protocol

AT A GLANCE

Protocol Summary

Measuring protease activity in test samples (Protocol A)

Prepare protease substrate solution (50 µL)
Add substrate control, positive control or test samples (50 µL)
Skip incubation for kinetic reading or incubate for 30 to 60 minutes for end point reading
Monitor fluorescence intensity at Ex/Em = 490/525 nm

Protocol Summary

Screening protease inhibitors using a purified enzyme (Protocol B)

Prepare protease substrate solution (10 µL)
Add substrate control, positive control, vehicle control or test samples (90 µL)
Skip incubation for kinetic reading or incubate for 30 to 60 minutes for end point reading
Monitor fluorescence intensity at Ex/Em = 490/525 nm

Important Thaw all the kit components at room temperature before starting the experiment. Please choose Protocol A or Protocol B according to your needs.

PREPARATION OF WORKING SOLUTION

1. Protease substrate solution (For protocol A)

Dilute Protease Substrate (Component A) at 1:100 in 2X assay buffer (Component C). Use 50 µL of protease substrate solution per assay in a 96-well plate.
Note The 2X Assay Buffer (Component C) is designed for detecting the activity of chymotrypsin, trypsin, thermolysin, proteinase K, protease XIV, and human leukocyte elastase. For other proteases, please refer to Table 1 below for the appropriate assay buffer formula.

2. Trypsin dilution (For protocol A)

Dilute Trypsin (5 U/µL, Component B) at 1:50 in de-ionized water to get a concentration of 0.1 U/µL.

3. Assay Buffer (1X) (For protocol B)

Add 5 mL de-ionized water into 5 mL of 2X Assay Buffer (Component C).

4. Protease substrate solution (For protocol B)

Dilute Protease Substrate (Component A) at 1:20 in 1X assay buffer. Use 10 µL/well of protease substrate solution for a 96-well plate.
Note The 2X assay buffer (Component C) is designed for detecting the activity of chymotrypsin, trypsin, thermolysin, proteinase K, protease XIV, and human leukocyte elastase. For other proteases, please refer to Table 1 below for the appropriate assay buffer formula.

5. Protease dilution (For protocol B)

Dilute the protease in 1X assay buffer to a concentration of 500 - 1000 nM (For Trypsin 50-100 U/mL). Each well will need 10 µL of protease dilution. Prepare an appropriate amount for all the test samples and extra for the positive control and vehicle control wells.
Table 1.Assay buffer formulas for proteases. For protocol A, 2X assay buffer is needed. For protocol B, 1X assay buffer is needed.

Protease	1X Assay Buffer
Cathepsin D	20 mM Sodium Citrate, pH 3.0
Papain	20 mM sodium acetate, 20 mM cysteine, 2 mM EDTA, pH 6.5
PAE	20 mM sodium phosphate, pH 8.0
Pepsin	10 mM HCl, pH 2.0
Porcine pancreas elastase	10 mM Tris-HCl, pH 8.8
Subtilisin	20 mM potassium phosphate buffer, pH 7.6, 150 mM NaCl

SAMPLE EXPERIMENTAL PROTOCOL

Protocol A: Measure protease activity in test samples

Table 1.Layout of the substrate control, positive control, and test samples in a 96-well microplate. SC=Substrate Control, PC =Positive Control, TS=Test Samples.

SC	SC	...	...
PC	PC	...	...
TS	TS
...	...

Table 2.Reagent composition for each well. If less than 50 µL of proteasecontaining biological sample is used, add ddH₂O to make a total volume of 50 µL.

Well	Volume	Reagent
SC	50 µL	De-ionized water
PC	50 µL	Trypsin dilution
TS	50 µL	Protease-containing solution

Add 50 µL of protease substrate solution (Protocol A) to all the wells in the assay plate. Mix the reagents well.
Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 490/525 nm. For kinetic reading: Immediately start measuring fluorescence intensity continuously and record data every 5 minutes for 30 minutes. For end-point reading: Incubate the reaction at a desired temperature for 30 to 60 minutes, protected from light. Then measure the fluorescence intensity.

Protocol B: Screening protease inhibitors using a purified enzyme

Table 3.Layout of the samples in a 96-well microplate. SC=Substrate Control, PC= Positive Control, VC=Vehicle Control, TS=Test Samples. It’s recommended to test at least three different concentrations of each test compound. All the test samples should be done in duplicates or triplicates.

SC	SC	...	...
PC	PC	...	...
VC	VC
TS	TS
...	...

Table 4.Reagent composition for each well. For each volume of test compound added into a well, the same volume of solvent used to deliver test compound needs to be checked for the effect of vehicle on the activity of protease.

Well	Volume	Reagent
SC	90 µL	Assay Buffer (1X) (90 µL)
PC	90 µL	Assay Buffer (1X) (80 µL) Protease dilution (10 µL)
VC	90 µL	Vehicle (X µL) Assay Buffer (80 - X µL) Protease dilution (10 µL)
TS	90 µL	Test compound (X µL) Assay Buffer (1X) (80 - X µL) Protease dilution (10 µL)

Add 10 µL of protease substrate solution (Protocol B) into the wells of positive control (PC), vehicle control (VC), and test sample (TS). Mix the reagents well.
Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em = 490 /525 nm. For kinetic reading: Immediately start measuring fluorescence intensity continuously and record data every 5 minutes for 30 minutes. For end-point reading: Incubate the reaction at a desired temperature for 30 to 60 minutes, protected from light. Then measure the fluorescence intensity.

Spectrum

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Product family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Correction Factor (260 nm)	Correction Factor (280 nm)
Amplite® Universal Fluorimetric Protease Activity Assay Kit Red Fluorescence	552	578	90000	0.32	0.178

Citations

View all 12 citations: Citation Explorer

Genetically programmed engineered living materials as high-performance bioplastics
Authors: Altin-Yavuzarslan, Gokce and Drake, Kinsey and Yuan, Shuo-Fu and Brooks, Sierra M and Kwa, Eng and Alper, Hal S and Nelson, Alshakim
Journal: Matter (2024)

Autophagy and UPS pathway contribute to nicotine-induced protection effect in Parkinson’s disease
Authors: Ullah, Inam and Uddin, Shahab and Zhao, Longhe and Wang, Xin and Li, Hongyu
Journal: Experimental Brain Research (2024): 1--16

Comparative assessment of commercially available wound gels in ex vivo human skin reveals major differences in immune response-modulatory effects
Authors: Seiser, S and Cerbu, D and Gallhofer, A and Matiasek, J and Elbe-B{\"u}rger, A
Journal: Scientific reports (2022): 1--9

Optimization of Isolation Method for Extracellular Vesicles from Pancreatic Juice and Impact of Protease Activity
Authors: Tsutsumi, Koichiro and Ueta, Eijiro and Kato, Hironari and Matsumoto, Kazuyuki and Horiguchi, Shigeru and Okada, Hiroyuki
Journal: Digestive diseases and sciences (2022): 1--8

A novel polyethylene glycol (PEG)-drug conjugate of Venetoclax, a Bcl-2 inhibitor, for treatment of acute myeloid leukemia (AML)
Authors: Ando, Hidenori and Murakami, Yuta and Eshima, Kiyoshi and Ishida, Tatsuhiro
Journal: Cancer Reports (2021): e1485

References

View all 30 references: Citation Explorer

Transient kinetic experiments demonstrate the existence of a unique catalytic enzyme form in the peptide-stimulated ATPase mechanism of Escherichia coli Lon protease
Authors: Vineyard D, Zhang X, Lee I.
Journal: Biochemistry (2006): 11432

Highly stable glycosylated serine protease from the medicinal plant Euphorbia milii
Authors: Yadav SC, P and e M, Jagannadham MV.
Journal: Phytochemistry (2006): 1414

Effects of Pseudomonas fluorescens M3/6 bacterial protease on plasmin system and plasminogen activation
Authors: Frohbieter KA, Ismail B, Nielsen SS, Hayes KD.
Journal: J Dairy Sci (2005): 3392

Fibrillar amyloid beta-protein inhibits the activity of high molecular weight brain protease and trypsin
Authors: Chauhan V, Sheikh AM, Chauhan A, Spivack WD, Fenko MD, Malik MN.
Journal: J Alzheimers Dis (2005): 37

Characterization of a novel and specific inhibitor for the pro-apoptotic protease Omi/HtrA2
Authors: Cilenti L, Lee Y, Hess S, Srinivasula S, Park KM, Junqueira D, Davis H, Bonventre JV, Alnemri ES, Zervos AS.
Journal: J Biol Chem (2003): 11489

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