Casein, FITC-conjugated

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<p>Proteases hydrolyze the quenching effect of the labeled 5-FITC, resulting in a bright green fluorescence proportional to protease activity.</p>
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5 mg 13440 $195


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Overview

Ex/Em (nm)494/521
SolventWater
Storage Freeze (<-15 °C)
Minimize light exposure
Category Enzyme Detection
Peptidases and Proteases
Related Secondary Reagents
Casein is considered to be a generic substrate for a broad spectrum of proteases. As native casein this fluoresceinated casein is hydrolyzed by many proteases, and widely used for fluorimetric measurement of protease activity. In the intact substrate, casein is heavily labeled with 5-FITC, resulting in significant fluorescence quenching. Protease-catalyzed hydrolysis relieves its quenching effect, yielding brightly green fluorescent dye-labeled short peptides. The increase in fluorescence intensity is directly proportional to protease activity. We do not recommend that this conjugate be used for fluorescence polarization assay. For fluorescence polarization we can custom-make the lightly labeled fluorescein casein conjugate.




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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

Chemical Properties of Casein
Appearance: Light yellow powder
Excitation/Emission: 494/521 nm
Solvent: Water

Preparation of stock solution
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Casein, FITC-conjugated stock solution:
Make a 5 - 10 mg/mL Casein, FITC-conjugated stock solution in PBS buffer.

Preparation of working solution

Casein FITC-conjugated working solution (2X):
Dilute the FITC-conjugated stock solution into 50 - 100 mM Tris buffer (pH 7.4) at 100 - 400 μg/mL. The 2X Assay working solution 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. The optimum concentration of the assay working solution should be determined experimentally for individual proteases.

Sample experimental protocol
  1. Mix equal volume of the trypsin standards or samples with 2X Assay working solution.

  2. Monitor the fluorescence increase at Ex/Em = 490/525 nm.

    a. For kinetic reading: Immediately start measuring fluorescence intensity continuously and record data every 5 minutes for 30 minutes.

    b. For end-point reading: Incubate the reaction at a desired temperature for 30 to 60 minutes, protected from light. Then measure the fluorescence intensity.

Table 1. Appropriate assay buffer formula for Assay working solution.

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
Example data analysis and figures

Figure 1.

Proteases hydrolyze the quenching effect of the labeled 5-FITC, resulting in a bright green fluorescence proportional to protease activity.

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

Liquid temperature measurement method in microchannels by using fluorescence polarization
Authors: Kazuya Tatsumi, Chi Hsuan Hsu, Atsushi Suzuki, Kazuyoshi Nakabe
Journal: Heat and Mass Transfer (2017): 1--10

Micro-scale temperature measurement method using fluorescence polarization
Authors: K Tatsumi, CH Hsu, A Suzuki, K Nakabe
Journal: (2016): 032097

Microscopic Fluid Temperature Measurements Using Fluorescence Polarization Method
Authors: Kazuya Tatsumi, Akihisa Tozaki, Kazuyoshi Nakabe
Journal: (2011): T10167--T10167






Additional Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Enzyme Probes & Assay Kits

Certificate of Analysis