Nbt Bcip Detection
Nbt Bcip Detection - Related Videos
04:54
Cell-Based AAV Neutralizing Antibody Assay: A Colorimetric Technique to Detect Neutralizing Antibodies Against Specific Adeno-Associated Viruses in Serum
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In this video, we demonstrate an in vitro colorimetric assay to detect the presence of neutralizing antibodies against alkaline phosphatase-expressing recombinant adeno-associated virus serotype 6 (rAAV6) vectors in sheep serum samples. The assay utilizes the reaction between the AAV encoding a human placental alkaline phosphatase gene and its substrate to generate an insoluble purple product.
04:46
In Situ Detection of Bacteria in Mouse Gingival Tissue Sections
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Source: Choi, Y. S., et al. In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA. J. Vis. Exp. (2015).This video demonstrates the in situ detection of bacteria in mouse gingival tissue sections using digoxin-labeled DNA probes targeting bacterial DNA. The section is incubated with the probe. Alkaline phosphatase-conjugated antibodies specific to digoxin are added. Endogenous phosphatases are inactivated, and a colored precipitate is...
04:20
Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay
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This video demonstrates the use of the ELISpot assay to assess the function of antibody-secreting B cells, ASCs. The antibodies secreted by the ASCs interact with the membrane-bound polyclonal antibody fragments, which are detected by the enzyme-conjugated IgG or IgM antibodies. The colored spot produced in each well indicates the presence of the specific antibody's isotype, which correlates with the B cells' secretory activity.
09:10
Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce
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Cited by 17 •
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09:00
Detection of Protein Ubiquitination
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Cited by 96 •
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Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.
FAQs
The reaction proceeds in linked stages. Alkaline phosphatase first hydrolyzes BCIP, generating an indoxyl intermediate. That intermediate is then oxidized, while NBT participates in the associated reduction chemistry that produces an insoluble blue-purple precipitate. Because formation depends on enzyme activity, the deposited color marks where the alkaline phosphatase signal is present, connecting molecular labeling to a visible biological readout.
The insoluble product stays at the target site rather than dispersing through the sample. This localization makes the method useful for spatial analysis, because color can be associated with a particular protein, nucleic acid, or cell location. It therefore helps distinguish where a biological signal occurs, not merely whether an assay contains enzyme-linked activity.
Alkaline phosphatase acts as the catalytic link between an enzyme-linked label and visible signal. It hydrolyzes BCIP to create an indoxyl intermediate, while NBT participates in the reaction that converts this chemistry into blue-purple insoluble product. Consequently, signal formation depends on activity at the labeled target, helping localize the detected biological component.
The assay format determines what biological pattern the color represents. In immunoblotting, it can identify labeled proteins; in situ hybridization uses the readout to visualize labeled nucleic acids; and tissue staining applies it to labeled cells. This makes the same chemistry adaptable to molecular and cellular localization questions.
At a basic level, the workflow links a biological target to alkaline phosphatase activity, exposes the reaction to BCIP and NBT, and then examines the resulting deposit at the target site. In immunoblotting, in situ hybridization, or tissue staining, this sequence converts an enzyme-linked signal into a localized visual pattern.
A blue-purple deposit indicates that the reaction has generated product at the target site. Interpreting the result focuses on location and pattern, allowing qualitative comparison of biological expression patterns. The readout is therefore especially informative when spatial distribution matters, such as in tissue staining or in situ hybridization.