Fluorogenic Substrate Hydrolysis
Fluorogenic Substrate Hydrolysis - Related Videos
03:05
Fluorescent Peptide Zymography: A Modified Technique to Detect Protease Activity Using Fluorogenic Substrates in Zymogram Gels
0 Views •
2025
In this video, we perform zymography, an electrophoretic technique, to analyze the proteolytic activity within biological samples.
04:10
Hydrolysis of an Ester - Concepts
0 Views •
2020
Triglycerides Triglycerides are triester molecules composed of a glycerol backbone that is bound to 3 fatty acids. These form the basis of many biological lipids that are found in vegetable oils and fats. Lipids that are isolated from plant material are the basis of vegetable oils, whereas lipids extracted from animals are used as lard or general sources of fats. A fatty acid molecule has a long carbon chain — between 12 and 20 carbons — with a weak organic acid at one end. Some fatty acids...
11:04
Hydrolysis of an Ester - Student Protocol
0 Views •
2020
Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA Preparation of SoapExpand Soap is prepared using a saponification reaction, where a base catalyzes the hydrolysis of three ester groups of an oil, such as coconut oil. During saponification, hydroxide ions from the base attack the carbonyl group on the oil to form a ratio of three molecules of soap to one molecule of glycerol. The resulting soap molecule is a long carbon chain, which is...
01:08
Hydrolysis of ATP
0 Views •
2019
The bonds of adenosine triphosphate (ATP) can be broken through the addition of water, releasing one or two phosphate groups in an exergonic process called hydrolysis. This reaction liberates the energy in the bonds for use in the cell—for instance, to synthesize proteins from amino acids. If one phosphate group is removed, a molecule of ADP—adenosine diphosphate—remains, along with inorganic phosphate. ADP can be further hydrolyzed to AMP—adenosine monophosphate—by the removal of a second...
08:11
Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers
0 Views •
Cited by 3 •
2022
The protocol presents two methods to determine the kinetics of the fluorogenic RNA aptamers Spinach2 and Broccoli. The first method describes how to measure fluorogenic aptamer kinetics in vitro with a plate reader, while the second method details the measurement of fluorogenic aptamer kinetics in cells by flow cytometry.
FAQs
Signal generation depends on the fluorophore’s chemical state before and after cleavage. In a substrate, the fluorophore is weakly fluorescent or nonfluorescent; enzyme-mediated bond cleavage separates or modifies it, increasing fluorescence under the assay conditions. This conversion links a molecular reaction to a measurable optical signal.
Substrate design determines which hydrolase activity is reported. A chemical bond must be compatible with the target enzyme, so protease, glycosidase, esterase, and other hydrolase assays use chemically distinct substrates. Specificity helps associate the fluorescence change with the intended reaction, while the selected assay conditions determine how clearly that change can be measured.
Fluorescence should be interpreted as a reaction-associated readout rather than an automatic direct measurement of enzyme amount. The signal often correlates with reaction progress or enzyme abundance, meaning the relationship depends on the defined assay conditions and the stage of the reaction. This distinction matters when comparing biological samples or treatment groups.
An assay workflow begins by pairing the target hydrolase with a substrate whose cleavable bond and fluorophore arrangement can report that activity. The reaction is then run under defined conditions, and fluorescence is measured with a plate reader, fluorescence microscope, or flow cytometer. The selected platform should match the biological system and intended measurement format.
A plate reader, fluorescence microscope, and flow cytometer can each measure the generated fluorescence, but they support different experimental formats. Selecting among them lets investigators adapt the readout to the biological system being studied, whether the work focuses on a biochemical assay, a cellular sample, or another defined measurement context.
Because the readout can support sensitive and quantitative measurements, this method is relevant to diagnostics, cell biology, drug screening, and biochemical research. Its scope extends across proteases, glycosidases, esterases, and other hydrolases, allowing investigators to connect fluorescence with different enzyme classes. The strategy therefore supports both mechanistic studies and applied screening contexts.