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

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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.
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500 Tests 13501 $195


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Overview

Ex/Em (nm)545/576
SolventDMSO
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsFluorescence microplate reader
Category Enzyme Detection
Peptidases and Proteases
Related Microplate Readers
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.




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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
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.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:540 nm
Emission:590 nm
Cutoff:570 nm
Recommended plate:Solid black
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. 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.

Protease 1X or 2X 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: 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. 

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

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.

Well Volume Reagent
SC 50 µL De-ionized water
PC 50 µL Typsin dilution
TS 50 µL Protease-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.

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

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.

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 (1X) (80 µL)
Protease dilution (10 µL)

TS 90 µL Test 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.
Example data analysis and figures

The fluorescence in the substrate control wells are used as a control, and subtracted from the values of sample wells with the enzymatic reactions.

Plot data in the format of relative fluorescence unit (RFU) versus time for each sample (as shown in Figure 1).

Determine the range of initial time points during which the reaction is linear. 10 - 15% conversion appears to be the optimal range.

Obtain the initial reaction velocity (Vo) in RFU/min. Determine the slope of the linear portion of the data plot.

A variety of data analyses can be run, e.g., determining inhibition %, IC50, Km, Ki, etc.

Figure 1. 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.
Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

Eosinophil extracellular trap cell death--derived DNA traps: Their presence in secretions and functional attributes
Authors: Shigeharu Ueki, Yasunori Konno, Masahide Takeda, Yuki Moritoki, Makoto Hirokawa, Yoshinori Matsuwaki, Kohei Honda, Nobuo Ohta, Shiori Yamamoto, Yuri Takagi
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: Junichi Kumamoto, Moe Tsutsumi, Makiko Goto, Masaharu Nagayama, Mitsuhiro Denda
Journal: Archives of dermatological research (2016): 49--54

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

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






Additional Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Enzyme Probes & Assay Kits

Application Notes
1. AssayWise Letters 2012, Vol 1(1)

Certificate of Analysis