Cellular Thermal Shift
Cellular Thermal Shift - Related Videos
03:09
Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O
0 Views •
Cited by 2 •
2024
Here, we present the thermal shift assay, a high-throughput, fluorescence-based technique used to investigate the binding of small molecules to proteins of interest.
09:42
Analysis of Thermal Expansion via Dilatometry
0 Views •
2023
Source: J. Jacob Chavez, Ryan T. Davis, and Taylor D. Sparks, Department of Materials Science and Engineering, The University of Utah, Salt Lake City, UT Thermal expansion is extremely important when considering which materials will be used in systems that experience fluctuations in temperature. A high or low thermal expansion in a material may or may not be desirable, depending on the application. For instance, in a common liquid thermometer, a material with a high thermal expansion would be...
09:12
Sound Waves and Doppler Shift
0 Views •
2023
Source: Arianna Brown, Asantha Cooray, PhD, Department of Physics & Astronomy, School of Physical Sciences, University of California, Irvine, CA Waves are disturbances that propagate through a material medium or empty space. Light waves can travel through a vacuum and some forms of matter, and are transverse in nature, which means that the oscillations are perpendicular to the direction of propagation. However, sound waves are pressure waves that travel through an elastic medium like air,...
06:57
Electrophoretic Mobility Shift Assay (EMSA)
0 Views •
2023
The electrophoretic mobility shift assay (EMSA) is a biochemical procedure used to elucidate binding between proteins and nucleic acids. In this assay a radiolabeled nucleic acid and test protein are mixed. Binding is determined via gel electrophoresis which separates components based on mass, charge, and conformation. This video shows the concepts of EMSA and a general procedure, including gel and protein preparation, binding, electrophoresis, and detection. Applications covered in this video...
07:22
How to Stabilize Protein: Stability Screens for Thermal Shift Assays and Nano Differential Scanning Fluorimetry in the Virus-X Project
0 Views •
Cited by 29 •
2019
A protocol is presented to rapidly test the thermal stability of proteins in a variety of conditions through thermal shift assays and nano differential scanning fluorimetry. Buffer systems, salts and additives, together comprising three unique stability screens, are assayed with proteins to identify suitable buffers for functional and structural studies.
FAQs
Heating creates a controlled stress that separates proteins by thermal behavior. Unbound proteins unfold and aggregate as temperature rises, reducing their representation in the soluble fraction, whereas ligand-bound proteins may remain soluble. The difference between treated and untreated samples across the temperature range therefore provides evidence that compound binding altered the stability of a cellular protein, rather than merely indicating compound presence.
An untreated sample provides the reference thermal behavior needed to interpret the treated sample. Researchers can examine how the soluble protein fraction changes across matching temperatures and determine whether treatment is associated with altered stability. This paired design helps distinguish a compound-related thermal effect from the unfolding and aggregation that occur during heating itself.
After heating, the assay focuses on the proteins that remain soluble rather than the aggregated material. Immunoblotting or mass spectrometry can quantify that soluble fraction, allowing researchers to track thermal stability changes for proteins of interest. The resulting measurements connect the physical heating response to evidence of compound engagement in the cellular sample.
Samples must include treated and untreated cells exposed to a temperature range, followed by measurement of the soluble fraction. Matching temperature conditions between samples is essential because interpretation depends on comparing their protein stability profiles. Researchers then use immunoblotting or mass spectrometry to quantify the retained soluble material and relate it to compound-associated changes.
Each compound can be evaluated by examining how treatment changes protein thermal stability relative to an untreated sample. Compounds that produce different stability patterns may show different degrees or profiles of target engagement. In drug discovery, these comparisons help prioritize activity while keeping the assessment tied to proteins in a native cellular environment.
Target engagement does not have to be assessed only for the intended protein. By measuring soluble-protein changes associated with treatment, researchers can look for interactions involving other cellular proteins as well. This makes the approach useful for identifying potential off-target interactions during drug discovery and for judging whether observed compound effects extend beyond the desired target.
Changes in protein stability measured after compound treatment can provide a cellular readout of drug action. These changes may help identify potential pharmacodynamic biomarkers, which are measurements related to how a compound affects its biological system. Such markers can connect molecular target engagement with compound treatment in cells, supporting medicine-focused drug discovery.