Navigation Links
Fluorometric Protease Assays in the SpectraMax Gemini Microplate Spectrofluorometer: Example Using Caspase3 (MaxLine Application Note #35)

Evelyn McGown, Ph.D. and Anna Lam, B.S.
Molecular Devices Corporation, 8/99


INTRODUCTION
Modern assays for proteolytic activity typically use synthetic chromogenic or flu-orogenic peptide substrates. Enzyme selectivity is obtained by choosing a peptide sequence uniquely recognized by the catalytic site of a particular enzyme. The chromophore or fluorophore is attached near the peptides cleavage site and enzymatic activity is detected by the increase in color or fluorescence. Chromogenic assays are easy in that one simply monitors the rise in absorbance above a (usually) low background. Fluorometric assays are much more sensitive than chromogenic assays, but are less straightforward. The substrate itself can be fluorescent, causing a high background. If the absorption spectra of the substrate and product overlap, the substrate can quench the signal from the product by absorbing either excitation or emission light. Therefore, both the excitation and emission wavelengths should be optimized to minimize interference from the substrate, while maintaining sufficiently high product fluorescence. This application note describes how to optimize a protease assay in the SPECTRAmax GEMINI micro-plate spectrofluorometer. We chose to use the protease caspase3 and a fluorogenic peptide substrate containing a coumarin derivative.

Coumarin derivatives are among the most popular fluorophores for peptide based protease assays because they are more sensitive and more watersoluble than other fluorescent derivatives.13 A 7amino4methylcoumarin (AMC) derivative was first used as a substrate for chymotrypsin1. Since then, AMC and the 7amino4trifluoromethylcoumarin (AFC) fluorophores have been used for various protease assays.14 In each case, cleavage of the fluorophore results in increased fluorescence, with a shift of excitation and emission maxima to longer wavelengths. AMC a
'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. Fluorescence-Based Single-Tube Assays to Rapidly Detect Human Gene Mutations
2. Matched siRNAs and Assays: Ambion + Applied Biosystems = RNAi Success
3. Using Validated siRNAs in Functional Genomic Assays
4. Nuclease Protection Assays: The Basics
5. Selection of Standards for Bio-Plex Cytokine Assays, Rev A
6. Feasibility of Multiplexing Bio-Plex Total Target and Phosphoprotein Assays
7. The Gradient Feature: Use in Optimization of Allelic Discrimination Assays, Rev B
8. The iCycler iQ Detection System for TaqMan Assays, Rev B
9. Multiplexed Quantitative Peptide Assays for Protein Biomarkers of Cardiovascular Disease in Human Plasma
10. Caspase-3 Activation - An Indicator of Apoptosis in Image-Based Assays
11. Amplification Efficiency of TaqMan Gene Expression Assays
Post Your Comments:
(Date:5/26/2015)... 26, 2015 WORMS, GERMANY – ... Co. (NYSE: GRA) announces that its manufacturing facility ... (GMP) certification from EXCiPACT™, an independent organization that ... , All three Grace facilities that ... excipient silica gels have now received GMP certification, ...
(Date:5/26/2015)... , Kalifornien und HILDEN, Deutschland, May ... QIAGEN Clinical Insight ® ... Genvarianten und die Erstellung dazugehöriger Berichte; Vorstellung ...     QIAGEN N.V. (NASDAQ: ... heute die Markteinführung von QIAGEN Clinical Insight ...
(Date:5/26/2015)... 2015  Sangamo BioSciences, Inc. (NASDAQ: SGMO ) announced ... will provide an update on the progress of Sangamo,s ZFP ... company,s business strategy at 1:30pm ET on Monday, June 1, ... is being held in New York, NY ... may be accessed via a link on the Sangamo BioSciences ...
(Date:5/26/2015)... , May 26, 2015  Alere Inc. (NYSE: ALR ... its failure to timely file its Quarterly Report on Form ... "Form 10-Q"), it has received a notice from the New ... in compliance with the NYSE,s continued listing requirements under the ... Listed Company Manual. The reason for the ...
Breaking Biology Technology:CORRECTION: Grace European Facility Receives GMP Excipient Certification for SYLOID® FP Silica Gel 2QIAGEN bringt klinische Bioinformatik-Plattform für Next Generation Sequencing-Anwendungen auf den Markt 2QIAGEN bringt klinische Bioinformatik-Plattform für Next Generation Sequencing-Anwendungen auf den Markt 3QIAGEN bringt klinische Bioinformatik-Plattform für Next Generation Sequencing-Anwendungen auf den Markt 4QIAGEN bringt klinische Bioinformatik-Plattform für Next Generation Sequencing-Anwendungen auf den Markt 5QIAGEN bringt klinische Bioinformatik-Plattform für Next Generation Sequencing-Anwendungen auf den Markt 6Sangamo BioSciences Announces Presentation At The Jefferies 2015 Global Healthcare Conference 2
... New observations could improve industrial production of high-quality ... to University of Illinois engineers. By combining data ... quality of graphene depends on the crystal structure of ... and computer engineering professors Joseph Lyding and Eric Pop, ...
... 2011 When we speak of exports, we ... however, technology, videogames or engineering, for example, also go out ... development.  This is the behavior shown by countries such as ... the leading countries in the export of services. Chile also ...
... SILVER SPRING, Md., Oct. 27, 2011 United Therapeutics ... financial results for the third quarter ended September 30, ... revenues this quarter, which reflects the continued strength of ... Chairman and Chief Executive Officer. "I,m also pleased that ...
Cached Biology Technology:Research: Graphene grows better on certain copper crystals 2Research: Graphene grows better on certain copper crystals 3Chile Exports 14% of its Services to the United States and Operates as a Nearshore Center for the U.S. 2Chile Exports 14% of its Services to the United States and Operates as a Nearshore Center for the U.S. 3Chile Exports 14% of its Services to the United States and Operates as a Nearshore Center for the U.S. 4United Therapeutics Corporation Reports Third Quarter 2011 Financial Results 2United Therapeutics Corporation Reports Third Quarter 2011 Financial Results 3United Therapeutics Corporation Reports Third Quarter 2011 Financial Results 4United Therapeutics Corporation Reports Third Quarter 2011 Financial Results 5United Therapeutics Corporation Reports Third Quarter 2011 Financial Results 6United Therapeutics Corporation Reports Third Quarter 2011 Financial Results 7United Therapeutics Corporation Reports Third Quarter 2011 Financial Results 8
(Date:5/11/2015)... JOSE, Calif. , May 11, 2015 /PRNewswire/ ... developer of human interface solutions, today announced the ... Vice President and Chief Financial Officer, reporting to ... Ali replaces Synaptics, current Chief Financial Officer, ... December 2014. Mr. Ali brings ...
(Date:5/10/2015)... 11, 2015 Fingerprint Cards (FPC) has ... FPC1155 from the distributor World Peace Industrial Group (WPI), part ... Asia . Deliveries are planned to take ... by smartphone manufacturers in China . The ... revenue guidance of + 1 000 MSEK for 2015.   ...
(Date:5/6/2015)... AVIV, Israel , May 6, 2015 ... world,s most established helmet producer, announced today that they ... Smart Helmet, the world,s first bio-sensing cycling helmet and ... be released in two new colors in order to ... for the product. In addition, LifeBEAM and Lazer announced ...
Breaking Biology News(10 mins):Synaptics Appoints Wajid Ali as Senior Vice President and Chief Financial Officer 2Fingerprint Cards Receives Touch Fingerprint Sensor Order of SEK 235 Million 2LifeBEAM and Lazer Announce Expanded Partnership in Bio-Sensing Sports Gear 2LifeBEAM and Lazer Announce Expanded Partnership in Bio-Sensing Sports Gear 3
... U.S. and now extends to much of the rest of ... is more people worldwide eating the so-called Western diet, which ... (the type of fat found in vegetable oil), and added ... as well as the weight gain it often causes, contributes ...
... one treatment offers therapeutic potential for multiple conditions. However, ... MD, principal investigator for the Center for Perinatal Research ... at Nationwide Children,s Hospital, and her team have found ... or heparin-binding EGF-like growth factor. Having discovered ...
... Amsterdam, NL, August 19, 2013 A new study ... that Chinese herbal medicine Ji-Sui-Kang (JSK), given systemically for ... reduced tissue damage, and preserved the structure of neural ... data showing that JSK may first act to reduce ...
Cached Biology News:Hitting the gym may help men avoid diet-induced erectile dysfunction 2HB-EGF protects intestines from a variety of injuries, pair of studies suggests 2Novel Chinese herbal medicine JSK improves spinal cord injury outcomes in rats 2
Guanine nucleotide-binding protein G(k), alpha subunit...
Goat anti-VAV2 Class: Antibody Product Group: Miscellaneous Antibody...
... P7 D7 F5] to RYK ( ... Antigen: Synthetic ... to amino acids 462 - 475 ... Gene ID: 6259 ...
DAP12 Market Segment: Protein Kinases...
Biology Products: