Fluorescent Probe Ligation
Fluorescent Probe Ligation - Related Videos
07:14
Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes
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Cited by 4 •
2014
We present a new fluorescence technique for selective in situ labeling of active phagocytic cells, which clear off cell corpses in stroke. The approach is important for assessing brain reaction to ischemia because only a small proportion of phagocytes present in ischemic brain participate in clearance of cell death.
04:02
An Assay to Measure the Accumulation of a Fluorescent Antibiotic Probe in Bacterial Cells
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2025
This video demonstrates an assay to measure the accumulation of a fluorescent antibiotic probe in bacterial cells. Exposure to an efflux pump inhibitor disrupts the transmembrane proton gradient in bacterial cells, upon which a fluorophore-conjugated antibiotic is added. Inhibition of efflux pumps due to the disrupted proton gradient causes accumulation of the antibiotic inside the bacteria. Bacterial cells are lysed, and the fluorescence intensity is measured to confirm antibiotic accumulation.
07:34
DNA Ligation Reactions
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2023
In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond. An enzyme known as a ligase catalyzes the ligation reaction. In the cell, ligases repair single and double strand breaks that occur during DNA replication. In the laboratory, DNA ligase is used during molecular cloning to join DNA fragments of inserts with vectors – carrier DNA molecules that will replicate target fragments in host organisms. This video provides an...
10:59
Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)
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Cited by 9 •
2012
A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.
11:21
Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells
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Cited by 5 •
2010
This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.
FAQs
Specificity depends on two probes recognizing adjacent regions of the same target strand. Their correct pairing places the probe ends in the alignment required for ligase activity. If the sequence pairing or arrangement is not sufficiently accurate, the joining reaction does not proceed as intended. This links sequence recognition directly to whether a fluorescent product can form.
The ligase step adds a sequence-dependent checkpoint beyond probe binding. Fluorescent probes must not only recognize the target but also align appropriately so ligase can join them. Because the detectable product depends on successful joining, this mechanism can improve specificity when identifying pathogen-associated or disease-associated nucleic acid sequences in biological samples.
The method connects distinct nucleic acid recognition events with fluorescence-based detection. In a complex sample, probe pairs can be designed around different target sequences, allowing multiple sequence-specific signals to be assessed within the same analytical framework. This supports multiplexed analysis of pathogens, infectious nucleic acids, or genetic markers rather than limiting detection to a single molecular target.
An assay first brings fluorescently labeled probes into contact with the DNA or RNA target so they can hybridize to adjacent sequences. When the pairing and alignment are sufficiently accurate, ligase joins the probes. The resulting ligated probe is then assessed through its fluorescent signal, which indicates successful sequence-dependent recognition and ligation.
Researchers may use Fluorescent Probe Ligation when they need to identify pathogen-related DNA or RNA, infectious nucleic acids, or disease-associated genetic markers. Its value is greatest when sensitive detection and sequence specificity are important. Fluorescence-based readouts also make the approach relevant to analyses of complex biological samples encountered in immunology and infection studies.
The fluorescent readout indicates that probe recognition and ligation occurred for a designated nucleic acid sequence. Consequently, it can support identification of pathogen-associated sequences, infectious nucleic acids, or disease-related genetic markers. When several target sequences are examined through fluorescence-based analysis, the method can contribute to multiplexed molecular profiling in complex biological samples.