Casein, TAMRA-conjugated
Ordering information
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Additional ordering information
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Physical properties
Molecular weight | N/A |
Solvent | Water |
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-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
Storage | Freeze (< -15 °C); Minimize light exposure |
UNSPSC | 12352200 |
Overview | SDSProtocol |
See also: Proteases
Molecular weight N/A | 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 |
Casein is considered to be a generic substrate for a broad spectrum of proteases. As native casein this rhodaminated casein is hydrolyzed by many proteases, and widely used for fluorometric measurement of protease activity. In the intact substrate, casein is heavily labeled with TAMRA, resulting in significant fluorescence quenching. Protease-catalyzed hydrolysis relieves its quenching effect, yielding brightly orange fluorescent dye-labeled short peptides. The increase in fluorescence intensity is directly proportional to protease activity. Compared to FITC-labeled casein, this casein substrate has pH-independent fluorescence. This feature is more convenient for the assays that require low pH. 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
PREPARATION OF STOCK SOLUTIONS
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.
Note Unused stock solution can be divided into single use aliquots and stored at -20 °C, and avoid exposure to light.
Casein, TAMRA-conjugated stock solution
Make a 2.5 - 5 mg/mL Casein, TAMRA-conjugated stock solution in PBS buffer.Note Unused stock solution can be divided into single use aliquots and stored at -20 °C, and avoid exposure to light.
PREPARATION OF WORKING SOLUTION
Casein TAMRA-conjugated working solution (2X)
Dilute the TAMRA-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
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 |
- Mix equal volume of the trypsin standards or samples with 2X Assay working solution.
- Monitor the fluorescence increase at Ex/Em = 540/590 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.
Spectrum
Open in Advanced Spectrum Viewer
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 |
Product Family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (280 nm) |
Casein, FITC-conjugated | 491 | 516 | 73000 | 0.92 | 0.35 |
Images
Citations
View all 3 citations: Citation Explorer
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
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
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
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
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
Authors: Yadav SC, P and e M, Jagannadham MV.
Journal: Phytochemistry (2006): 1414
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
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
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
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
Measurement of protease activity of live Uronema marinun (Ciliata: Scuticociliatida) by fluorescence polarization
Authors: Lee EH, Kim CS, Cho JB, Ahn KJ, Kim KH.
Journal: Dis Aquat Organ (2003): 85
Authors: Lee EH, Kim CS, Cho JB, Ahn KJ, Kim KH.
Journal: Dis Aquat Organ (2003): 85
Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis
Authors: Rudyak SG, Brenowitz M, Shrader TE.
Journal: Biochemistry (2001): 9317
Authors: Rudyak SG, Brenowitz M, Shrader TE.
Journal: Biochemistry (2001): 9317
Directed polar secretion of protease from single cells of Vibrio cholerae via the type II secretion pathway
Authors: Scott ME, Dossani ZY, S and kvist M., undefined
Journal: Proc Natl Acad Sci U S A (2001): 13978
Authors: Scott ME, Dossani ZY, S and kvist M., undefined
Journal: Proc Natl Acad Sci U S A (2001): 13978
Evidence for tryptophan in proximity to histidine and cysteine as essential to the active site of an alkaline protease
Authors: Tanksale AM, Vernekar JV, Ghatge MS, Deshp and e VV., undefined
Journal: Biochem Biophys Res Commun (2000): 910
Authors: Tanksale AM, Vernekar JV, Ghatge MS, Deshp and e VV., undefined
Journal: Biochem Biophys Res Commun (2000): 910
Effect of psychrotrophic bacteria and of an isolated protease from Pseudomonas fluorescens M3/6 on the plasmin system of fresh milk
Authors: Fajardo-Lira C, Oria M, Hayes KD, Nielsen SS.
Journal: J Dairy Sci (2000): 2190
Authors: Fajardo-Lira C, Oria M, Hayes KD, Nielsen SS.
Journal: J Dairy Sci (2000): 2190
Application notes
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Restriction of Advanced Glycation End Products Improves Insulin Resistance in Human Type 2 Diabetes
Matrix Remodeling Maintains Embryonic Stem Cell Self-Renewal by Activating Stat3
Human High Temperature Requirement Serine Protease A1 (HTRA1) Degrades Tau Protein Aggregates
Selective Detection of Pyrophosphate Using a Fluorogenic Pyrophosphate Sensor
Restriction of Advanced Glycation End Products Improves Insulin Resistance in Human Type 2 Diabetes
Matrix Remodeling Maintains Embryonic Stem Cell Self-Renewal by Activating Stat3
Human High Temperature Requirement Serine Protease A1 (HTRA1) Degrades Tau Protein Aggregates
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