Amplite® Universal Fluorimetric Protease Activity Assay Kit Green Fluorescence

Trypsin protease activity was analyzed by Amplite® Universal Fluorimetric Protease Activity Assay Kit. Protease substrate was incubated with 1 unit trypsin in the kit assay buffer. The control wells had protease substrate only (without trypsin). The fluorescence signal was measured starting from time 0 when trypsin was added. Samples were done in triplicates.

Protease activity of keratinocytes treated with Cry j1 with or without tranexamic acid, SBTI and bivalirudin. (a) Application of Cry j1 (100 ng/ml) to cultured human keratinocytes at time 0 s induced a rapid, transient increase of protease activity (red line). After application of BSS(+) alone (control), the fluorescence level gradually decreased (gray line). The transient increase was blocked by tranexamic acid (blue line), SBTI (yellow line) or bivalirudin (purple line). The vertical scale is normalized by the fluorescence at time 0. (b) Quantitation of fluorescence change within 1 min after application (n = 30–50 cells). A significant difference was observed between the control and Cry j1 application groups. Tranexamic acid, SBTI and bivalirudin each blocked the increase. Similar results were obtained in three independent experiments. Bars and lines represent mean ± SD. (c) Application of Cry j1 (100 ng/ml) to the cells treated with scramble RNA at time 0 s induced a rapid, transient increase of protease activity (gray line), while the application of the same amount of Cry j1 to the thrombin siRNA-treated cells induced a low transient increase (cyan line). The vertical scale is normalized by the fluorescence at time 0. (d) Quantitation of fluorescence change within 1 min after application (n = 30–50 cells). A significant difference was observed between the thrombin siRNA-treated cells and control. Similar results were obtained in three independent experiments. Bars and lines represent mean ± SD. Source: <strong>Tranexamic acid blocks the thrombin-mediated delay of epidermal permeability barrier recovery induced by the cedar pollen allergen, Cry j1 </strong>by Nakanishi et al., <em>Scientific Reports</em>, Oct. 2018.

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Shipping	Standard overnight for United States, inquire for international

Spectral properties

Correction Factor (280 nm)	0.35
Extinction coefficient (cm ^-1 M ^-1)	73000
Excitation (nm)	491
Emission (nm)	516
Quantum yield	0.92

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Alternative formats

Amplite® Universal Fluorimetric Protease Activity Assay Kit *Red Fluorescence*

Overview SDS Protocol

Correction Factor (280 nm)

0.35

Extinction coefficient (cm ^-1 M ^-1)

73000

Excitation (nm)

491

Emission (nm)

516

Quantum yield

0.92

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.

Platform

Fluorescence microplate reader

Excitation	490 nm
Emission	525 nm
Cutoff	515 nm
Recommended plate	Solid black

Components

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

Open in Advanced Spectrum Viewer

Spectral properties

Correction Factor (280 nm)	0.35
Extinction coefficient (cm ^-1 M ^-1)	73000
Excitation (nm)	491
Emission (nm)	516
Quantum yield	0.92

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

Images

Figure 1. Trypsin protease activity was analyzed by Amplite® Universal Fluorimetric Protease Activity Assay Kit. Protease substrate was incubated with 1 unit trypsin in the kit assay buffer. The control wells had protease substrate only (without trypsin). The fluorescence signal was measured starting from time 0 when trypsin was added. Samples were done in triplicates.

Figure 2. Protease activity of keratinocytes treated with Cry j1 with or without tranexamic acid, SBTI and bivalirudin. (a) Application of Cry j1 (100 ng/ml) to cultured human keratinocytes at time 0 s induced a rapid, transient increase of protease activity (red line). After application of BSS(+) alone (control), the fluorescence level gradually decreased (gray line). The transient increase was blocked by tranexamic acid (blue line), SBTI (yellow line) or bivalirudin (purple line). The vertical scale is normalized by the fluorescence at time 0. (b) Quantitation of fluorescence change within 1 min after application (n = 30–50 cells). A significant difference was observed between the control and Cry j1 application groups. Tranexamic acid, SBTI and bivalirudin each blocked the increase. Similar results were obtained in three independent experiments. Bars and lines represent mean ± SD. (c) Application of Cry j1 (100 ng/ml) to the cells treated with scramble RNA at time 0 s induced a rapid, transient increase of protease activity (gray line), while the application of the same amount of Cry j1 to the thrombin siRNA-treated cells induced a low transient increase (cyan line). The vertical scale is normalized by the fluorescence at time 0. (d) Quantitation of fluorescence change within 1 min after application (n = 30–50 cells). A significant difference was observed between the thrombin siRNA-treated cells and control. Similar results were obtained in three independent experiments. Bars and lines represent mean ± SD. Source: Tranexamic acid blocks the thrombin-mediated delay of epidermal permeability barrier recovery induced by the cedar pollen allergen, Cry j1 by Nakanishi et al., Scientific Reports, Oct. 2018.

Citations

View all 10 citations: Citation Explorer

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

Chondroitin sulfate--mediated N-cadherin/$\beta$-catenin signaling is associated with basal-like breast cancer cell invasion
Authors: Nadanaka, Satomi and Kinouchi, Hiroki and Kitagawa, Hiroshi
Journal: Journal of Biological Chemistry (2018): 444--465

IL-33, IL-25 and TSLP contribute to development of fungal-associated protease-induced innate-type airway inflammation
Authors: Hiraishi, Yoshihisa and Yamaguchi, Sachiko and Yoshizaki, Takamichi and Nambu, Aya and Shimura, Eri and Takamori, Ayako and Narushima, Seiko and Nakanishi, Wakako and Asada, Yosuke and Numata, Takafumi and others, undefined
Journal: Scientific reports (2018): 18052

Tranexamic acid blocks the thrombin-mediated delay of epidermal permeability barrier recovery induced by the cedar pollen allergen, Cry j1
Authors: Nakanishi, S and Kumamoto, J and Denda, M
Journal: Scientific reports (2018): 15610

Eosinophil extracellular trap cell death--derived DNA traps: Their presence in secretions and functional attributes
Authors: Ueki, Shigeharu and Konno, Yasunori and Takeda, Masahide and Moritoki, Yuki and Hirokawa, Makoto and Matsuwaki, Yoshinori and Honda, Kohei and Ohta, Nobuo and Yamamoto, Shiori and Takagi, Yuri and others, undefined
Journal: Journal of Allergy and Clinical Immunology (2016): 258--267

Japanese Cedar (Cryptomeria japonica) pollen allergen induces elevation of intracellular calcium in human keratinocytes and impairs epidermal barrier function of human skin ex vivo
Authors: Kumamoto, Junichi and Tsutsumi, Moe and Goto, Makiko and Nagayama, Masaharu and Denda, Mitsuhiro
Journal: Archives of dermatological research (2016): 49--54

Impact of silk biomaterial structure on proteolysis
Authors: Brown, Joseph and Lu, Chia-Li and Coburn, Jeannine and Kaplan, David L
Journal: Acta biomaterialia (2015): 212--221

Incorporation of proteinase inhibitors into silk-based delivery devices for enhanced control of degradation and drug release
Authors: Pritchard, Eleanor M and Valentin, Thomas and Boison, Detlev and Kaplan, David L
Journal: Biomaterials (2011): 909--918

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
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Highly stable glycosylated serine protease from the medicinal plant Euphorbia milii
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Effects of Pseudomonas fluorescens M3/6 bacterial protease on plasmin system and plasminogen activation
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Fibrillar amyloid beta-protein inhibits the activity of high molecular weight brain protease and trypsin
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Characterization of a novel and specific inhibitor for the pro-apoptotic protease Omi/HtrA2
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Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis
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Directed polar secretion of protease from single cells of Vibrio cholerae via the type II secretion pathway
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Journal: Proc Natl Acad Sci U S A (2001): 13978

Effect of psychrotrophic bacteria and of an isolated protease from Pseudomonas fluorescens M3/6 on the plasmin system of fresh milk
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Expression, purification, and functional analysis of the human serine protease HtrA2
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