Fluorometric Substrate
Fluorometric Substrate - Related Videos
12:33
High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities
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Cited by 485 •
2013
To measure potential rates of soil extracellular enzyme activities, synthetic substrates that are bound to a fluorescent dye are added to soil samples. Enzyme activity is measured as the fluorescent dye is released from the substrate by an enzyme-catalyzed reaction, where higher fluorescence indicates more substrate degradation.
08:05
Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting
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Cited by 19 •
2011
Using Luminex Corporation’s xMAP microsphere technology, we have developed the Multiplexed Fluorometric ImmunoAssay (MFIA) for serosurveillance of various laboratory animal species. The MFIA is a suspension microarray where antigen, tissue control or immunoglobulins are covalently linked to color-coded polystyrene microspheres. The MFIA testing method as well as various troubleshooting topics is addressed.
05:29
Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry
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Cited by 6 •
2023
The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.
11:13
Identification of Kinase-substrate Pairs Using High Throughput Screening
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Cited by 1 •
2015
Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.
09:22
High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization
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Cited by 8 •
2014
Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.
FAQs
The key event is a reaction-dependent change in fluorescence. Hydrolysis, oxidation, or another specific transformation can convert a nonfluorescent or weakly fluorescent compound into a fluorophore, the light-emitting product. Measuring the resulting signal during a reaction provides a way to follow progress, while the chemical response preserves specificity for the transformation being investigated.
Substrate chemistry determines when the signal appears. A compound that becomes fluorescent after enzymatic hydrolysis can report activity associated with enzymes capable of carrying out that reaction. Oxidation or another transformation uses a different chemical route to produce the signal. Selecting the reaction therefore links fluorescence to the particular process being investigated.
Fluorescence intensity is useful because it changes as the reaction proceeds, but interpretation depends on keeping assay conditions controlled. Differences in signal can then be related to reaction progress rather than uncontrolled changes during measurement. This principle supports comparisons among environmental samples, including assessments of relative functional activity, rather than treating fluorescence as an unqualified biological measurement.
The measurement workflow centers on exposing a sample to the selected fluorometric substrate under controlled conditions, allowing the relevant reaction to occur, and recording fluorescence. Researchers can then use the signal to follow reaction progress or compare activity between samples. Because the assay can use low volumes, it fits analyses of water, soil, sediments, and wastewater.
Researchers use these assays to examine extracellular enzyme activity or microbial processes in environmental materials. Relevant settings include water, soil, sediments, and wastewater. The measurements can support studies of organic-matter decomposition and comparisons of functional activity across samples, making the method useful for connecting chemical reaction measurements with environmental biology.
Changes in fluorescence-derived activity can indicate biological responses to pollution or changing environmental conditions. Comparisons across samples may show that functional activity differs, while measurements of reaction progress provide a basis for evaluating the direction or extent of that difference. These results are most meaningful when assay conditions are controlled consistently across the samples being compared.
Low-volume fluorescence analyses make it possible to examine environmental samples without requiring large sample quantities. In work with water, soil, sediments, or wastewater, that format supports sensitive measurements of enzyme activity and microbial processes while keeping the assay compatible with limited-volume samples. The resulting data can be used to compare activity or evaluate responses to environmental change.