Dab Substrate Reaction
Dab Substrate Reaction - Related Videos
09:16
Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro
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Cited by 1 •
2014
RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.
07:20
Grignard Reaction
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2023
Source: Vy M. Dong and Faben Cruz, Department of Chemistry, University of California, Irvine, CA This experiment will demonstrate how to properly carry out a Grignard reaction. The formation of an organometallic reagent will be demonstrated by synthesizing a Grignard reagent with magnesium and an alkyl halide. To demonstrate a common use of a Grignard reagent, a nucleophilic attack onto a carbonyl will be performed to generate a secondary alcohol by forming a new C-C bond.
04:25
Substrate Oxidation Assay in Cultured Cells: An In Vitro Assay to Quantify Substrate Oxidation in Cells by Measuring Radioactive Signals from Trapped CO2
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2025
This video demonstrates a quantification technique for CO2 released during substrate oxidation using 14C-radiolabeled substrates. The released 14CO2 is trapped in an alkaline solution and quantified by a scintillation counter. The oxidation of different substrates varies between tissues and reflects the pathophysiological condition of the tissue.
11:53
Substrate Generation for Endonucleases of CRISPR/Cas Systems
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Cited by 4 •
2012
CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.
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.
FAQs
The signal forms through a coupled chemical sequence: hydrogen peroxide enables horseradish peroxidase to oxidize DAB, and the oxidized substrate accumulates as an insoluble brown product. Because deposition occurs where the enzyme is present, the reaction links enzyme distribution to a visible location in the section. This chemistry converts protein or antigen localization into a microscopy-readable tissue pattern.
Horseradish peroxidase acts as the catalytic component, hydrogen peroxide provides the oxidizing condition, and DAB serves as the substrate that becomes visibly deposited after oxidation. These components contribute different functions: the enzyme drives the reaction, peroxide supports oxidation, and DAB records the event as color. Separating these roles helps explain how staining reflects enzyme-associated localization.
The precipitate remains at the site where enzyme activity occurred rather than dispersing throughout the sample. This retention preserves spatial information, allowing observers to relate the brown signal to particular proteins, antigens, or cellular structures within a fixed section. Its stable staining product also supports examination and comparison under a light microscope after the reaction has occurred.
A useful reaction requires DAB, horseradish peroxidase, and hydrogen peroxide within a biological sample prepared as a fixed tissue section. The visible outcome depends on the enzyme being present where the target-associated signal is located, because oxidation and precipitate formation occur at those sites. Light microscopy then provides the means to examine the resulting brown pattern.
The brown deposit identifies where horseradish peroxidase-associated activity occurs in the sample. In immunohistochemistry and tissue staining, that location can be used to identify and map proteins, antigens, or cellular structures while retaining their position within the tissue. The pattern therefore supports spatial assessment of protein expression rather than providing only a signal from a dissolved or homogenized sample.
Its combination of spatial localization, a stable colored product, and compatibility with light microscopy makes the reaction useful for examining fixed biological samples. Applications include diagnostic histopathology, cell biology, immunohistochemistry, tissue staining, and experimental evaluation of protein expression. In each setting, the brown precipitate connects molecular or enzyme-associated activity with recognizable tissue architecture.