logo
AAT Bioquest

Amplite® Universal Fluorimetric Protease Activity Assay Kit *Red 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 the test sample (Protocol A)

  1. Prepare Protease Substrate working solution (50 µL)
  2. Add substrate control, positive control or test samples (50 µL)
  3. Skip incubation for kinetic reading or incubate 30 minutes - 1 hour for end point reading
  4. Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Protocol summary
Screening protease inhibitors using a purified enzyme (Protocol B)

  1. Prepare Protease Substrate working solution (10 µL)
  2. Add substrate control, positive control, vehicle control or test samples (90 µL)
  3. Skip incubation for kinetic reading or incubate 30 minutes -1 hour for end point reading
  4. Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
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

For Protocol A

1. Protease Substrate working solution (for Protocol A):
Dilute Protease Substrate (Component A) at 1:100 in 2X Assay Buffer (Component C) to make Protease Subtrate working solution for Protocol A. Use 50 µL/well of Protease Substrate working 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 for the appropriate assay buffer formula.

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

For Protocol B

1. Assay buffer (1X):
Add 5 mL of de-ionized water to 5 mL of 2X Assay Buffer (Component C) to make 1X Assay buffer.

2. Protease Substrate working solution (for Protocol B) :
Dilute Protease Substrate (Component A) at 1:20 in 1X Assay buffer to make Protease Substrabe working solution for Protocol B. Use 10 µL/well of Protease Substrate working 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 for the appropriate assay buffer formula.

3. Protease dilution:
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 diluent. 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.

Protease1X or 2X Assay Buffer
Cathepsin D20 mM Sodium Citrate, pH 3.0
Papain20 mM sodium acetate, 20 mM cysteine, 2 mM EDTA, pH 6.5
PAE20 mM sodium phosphate, pH 8.0
Pepsin10 mM HCl, pH 2.0
Porcine pancreas elastase10 mM Tris-HCl, pH 8.8
Subtilisin20 mM potassium phosphate buffer, pH 7.6, 150 mM NaCl

SAMPLE EXPERIMENTAL PROTOCOL

Protocol A: Measuring protease activity in test samples

Table 2A. Layout of the substrate control, positive control, and test samples in a solid black 96-well microplate. SC=Substrate Control, PC =Positive Control, TS=Test Samples. 

SCSC......
PCPC......
TSTS  
......  
......  
    
    
    

Table 3A. Reagent composition for each well. If less than 50 µL of protease-containing biological sample is used, add ddH2O to make a total volume of 50 µL.

WellVolumeReagent
SC50 µLDe-ionized water
PC50 µLTypsin dilution
TS50 µLProtease-containing samples
  1. Prepare Subtrate Control (SC), Positive Control (PC) and Test Samples (TS) according to the layout provided in Table 2A and Table 3A.

  2. Add 50 µL of Protease Substrate working solution (Protocol A) to all the wells in the assay plate. Mix the reagents well.

  3. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 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 2B. Layout of the samples in a solid black 96-well microplate.  SC=Substrate Control, PC= Positive Control, VC=Vehicle Control, TS=Test Samples. It is recommended to test at least three different concentrations of each test compound. All the test samples should be done in duplicates or triplicates.

SCSC......
PCPC......
VCVC  
TSTS  
......  
......  
    
    

Table 3B. 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.

WellVolumeReagent
SC90 µLAssay Buffer (1X) (90 µL)
PC

90 µL

Assay Buffer (1x) (80 µL)
Protease dilution (10 µL)

VC90 µL

Vehicle (X µL)
Assay Buffer (1X) (80 µL)
Protease dilution (10 µL)

TS90 µLTest compound (X µL)
Assay Buffer (1X) (80 -X µL)
Protease dilution (10 µL)
  1. Prepare Subtrate Control (SC), Positive Control (PC), Vehicle Control (VC) and Test Samples (TS) according to the layout provided in Table 2B and Table 3B.

  2. Add 10 µL of Protease Substrate working solution (Protocol B) into the wells of substrate control (PC), positive control (PC), vehicle control (VC), and test sample (TS) wells. Mix the reagents well.

  3. Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 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

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (280 nm)
Amplite® Universal Fluorimetric Protease Activity Assay Kit *Green Fluorescence*491516730000.920.35

Citations

View all 7 citations: Citation Explorer
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
Gene expression dynamics in input-responsive engineered living materials programmed for bioproduction
Authors: Sugianto, Widianti and Altin-Yavuzarslan, Gokce and Tickman, Benjamin I and Kiattisewee, Cholpisit and Yuan, Shuo-Fu and Brooks, Sierra M and Wong, Jitkanya and Alper, Hal S and Nelson, Alshakim and Carothers, James M
Journal: Materials Today Bio (2023): 100677
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
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

References

View all 30 references: Citation Explorer
Highly stable glycosylated serine protease from the medicinal plant Euphorbia milii
Authors: Yadav SC, P and e M, Jagannadham MV.
Journal: Phytochemistry (2006): 1414
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
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
Page updated on October 14, 2024

Ordering information

Price
Unit size
Catalog Number13501
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
Technical SupportContact us
Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Spectral properties

Correction Factor (260 nm)

0.32

Correction Factor (280 nm)

0.178

Extinction coefficient (cm -1 M -1)

90000

Excitation (nm)

552

Emission (nm)

578

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components

Trypsin protease activity was analyzed by using Amplite® Universal Fluorimetric Protease Activity Assay Kit. Protease substrate was incubated with 3 units of trypsin. 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 triplicate.
Trypsin protease activity was analyzed by using Amplite® Universal Fluorimetric Protease Activity Assay Kit. Protease substrate was incubated with 3 units of trypsin. 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 triplicate.
Trypsin protease activity was analyzed by using Amplite® Universal Fluorimetric Protease Activity Assay Kit. Protease substrate was incubated with 3 units of trypsin. 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 triplicate.