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Casein, FITC-conjugated

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.

Example protocol

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.

Protease1X 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

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Correction Factor (260 nm)Correction Factor (280 nm)
Casein, TAMRA-conjugated552578900000.320.178

Citations

View all 5 citations: Citation Explorer
Development of novel imipridone derivatives with potent anti-cancer activities as human caseinolytic peptidase P (hClpP) activators
Authors: Zhang, Yanzhi and Jiang, Jinxin and Ding, Hao and Li, Qiannan and Xiao, Yibei and Sun, Haiying
Journal: Bioorganic Chemistry (2024): 107765
Liquid temperature measurement method in microchannels by using fluorescence polarization
Authors: Tatsumi, Kazuya and Hsu, Chi Hsuan and Suzuki, Atsushi and Nakabe, Kazuyoshi
Journal: Heat and Mass Transfer (2018): 2607--2616
Liquid temperature measurement method in microchannels by using fluorescence polarization
Authors: Tatsumi, Kazuya and Hsu, Chi Hsuan and Suzuki, Atsushi and Nakabe, Kazuyoshi
Journal: Heat and Mass Transfer (2017): 1--10
Micro-scale temperature measurement method using fluorescence polarization
Authors: Tatsumi, K and Hsu, CH and Suzuki, A and Nakabe, K
Journal: (2016): 032097
Microscopic Fluid Temperature Measurements Using Fluorescence Polarization Method
Authors: Tatsumi, Kazuya and Tozaki, Akihisa and Nakabe, Kazuyoshi
Journal: (2011): T10167--T10167

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
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
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
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

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Catalog Number13440
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Additional ordering information

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Physical properties

Molecular weight

N/A

Solvent

Water

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

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200
Proteases hydrolyze the quenching effect of the labeled 5-FITC, resulting in a bright green fluorescence proportional to protease activity.
Proteases hydrolyze the quenching effect of the labeled 5-FITC, resulting in a bright green fluorescence proportional to protease activity.
Proteases hydrolyze the quenching effect of the labeled 5-FITC, resulting in a bright green fluorescence proportional to protease activity.