Small Molecule Assays
Small Molecule Assays - Related Videos
12:05
A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA
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Cited by 8 •
2017
This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.
10:04
A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity
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Cited by 13 •
2014
Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.
04:16
Cell Aggregation Assay to Detect Trans Interactions between Cell Adhesion Molecules
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2025
In this video, we demonstrate the technique to measure transcellular interactions between synaptic cell adhesion molecules through the HEK cell aggregation assay. In this assay, an equal number of HEK cells expressing a desired presynaptic cell adhesion molecule and a fluorescent protein are mixed with HEK cells expressing a post-synaptic cell adhesion molecule and another fluorescent protein. The interaction between the cell adhesion molecules forces the cells to aggregate, which can be...
08:43
A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules
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Cited by 10 •
2017
We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.
03:32
Amplified Luminescent Proximity Homogeneous Assay: A Bead-Based Proximity Assay to Screen Small Molecules Inhibiting Protein-Protein Interactions
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2025
In this video, we demonstrate the amplified luminescent proximity homogeneous assay—a bead-based proximity assay. It utilizes homogeneously-sized acceptor and donor beads, immobilized with two proteins that tend to interact to screen potential small molecules inhibiting protein interactions.
FAQs
The assay links a compound’s biological effect to an observable signal, such as protein binding, altered enzyme activity, fluorescence, or a cellular response. Researchers compare that signal with appropriate controls and across a concentration series. This design helps distinguish compound-dependent effects from background activity and supports estimates of potency, meaning the concentration associated with a defined response.
These formats measure different biological consequences. Binding assays examine interaction with a protein, whereas enzyme assays monitor changes in catalytic activity. Fluorescence-based formats use a measurable optical signal, and cellular assays assess responses in living cells. Comparing results across formats can help connect molecular interaction with functional effect, rather than relying on a single type of readout.
A concentration series shows how the measured response changes as compound exposure increases, allowing potency to be estimated rather than inferred from one observation. Selectivity compares activity across relevant targets or responses. Together, these measurements help determine whether a candidate produces a specific biological effect or broadly alters several systems, which is important when linking structure to function.
A basic workflow establishes controls, applies the compound across multiple concentrations, and records a defined measurable endpoint. The endpoint may be binding, enzyme activity, fluorescence, or a cellular response, depending on the assay format. Controls provide the comparison needed to interpret signal changes, while concentration-dependent results support characterization of activity and potency.
These assays support several stages of biological investigation, including drug discovery, target validation, metabolic studies, and toxicology. They can identify compounds that produce a desired response, test whether a proposed target is involved, examine effects on biological processes, and evaluate potentially harmful activity. Their value comes from connecting compound behavior with measurable biological function.
A positive result can identify an active candidate and provide evidence that the compound affects the measured biological system. When results are compared across assay types, concentrations, and targets, they may also reveal a possible mechanism of action or differences in selectivity. These findings can guide development of interventions designed to act more selectively.