Adapter Ligation Assay
Adapter Ligation Assay - Related Videos
04:49
Proximity Ligation Assay to Study In Situ Protein-Protein Interactions
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2025
This video describes the proximity ligation assay to study in situ protein-protein interactions in nematode cells. The close proximity of the proteins of interest generates the circular DNA template with the help of a PLA probe attached to a secondary antibody. The probe is further amplified using the amplification solution, which produces a long single-stranded DNA followed by the hybridization of the detector probe, enabling the localization of protein-protein interactions in cells.
03:08
Detection of Protein-Protein Interactions in Drosophila Larvae Using a Proximity Ligation Assay
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2025
This video demonstrates the visualization of protein-protein interactions at the Drosophila neuromuscular junction (NMJ) using Proximity Ligation Assay (PLA). After isolating the body walls, interacting proteins are probed with primary antibodies and PLA-conjugated secondary antibodies. The proximity of the interacting proteins facilitates the ligation of probes with connector oligonucleotides, forming circular DNA, which is then amplified by rolling circle amplification. Fluorescent probes...
09:34
Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells
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Cited by 59 •
2016
Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.
12:52
Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay
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Cited by 34 •
2011
Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.
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...
FAQs
The ligation step depends on chemical compatibility between the adapter ends and the DNA or RNA ends. If the target ends are not ready to join, preparation can include end repair or creation of defined overhangs. These preceding choices determine whether the ligase can connect the molecules efficiently and establish the intended adapter-target structure for later analysis.
Adapter design determines what can happen after ligation. A primer-binding site can support subsequent amplification, a sample identifier can distinguish material from different sources, and a capture sequence can help select molecules for analysis. Thus, the adapter is not merely a joining component; its sequence supplies functional information that shapes the downstream workflow.
Many DNA and RNA molecules do not begin with standardized sites that are convenient for amplification, identification, or analysis. Adding designed adapter sequences supplies those missing handles. This makes otherwise difficult molecules more accessible to common downstream operations and allows researchers to examine them within a consistent library or detection strategy.
A typical workflow first prepares the DNA or RNA ends, using end repair or defined overhang formation when needed. Designed adapters are then combined with the prepared molecules under conditions that allow a DNA or RNA ligase to join chemically compatible ends. The resulting products proceed to amplification, sequencing, or targeted detection, depending on adapter design.
In library preparation, adapters convert individual DNA or RNA molecules into analyzable library members by adding sequences that downstream processes can recognize. Their primer-binding sites can support amplification, while their compatibility with sequencing workflows enables molecule identification and analysis. This makes adapter ligation useful when starting molecules lack uniform sites required for library construction.
Targeted detection can use adapters as selective molecular handles rather than only as amplification sites. A capture sequence incorporated into the adapter provides a feature for focusing analysis on molecules carrying that sequence, while sample identifiers help distinguish sources. The resulting design supports organized identification of selected DNA or RNA molecules during downstream analysis.