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Related Concept Videos

Quantitative Analysis01:12

Quantitative Analysis

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Quantitative analysis is a technique for measuring the amount of specific constituents in a sample. When the sample's composition is unknown, qualitative analysis is performed first to identify its components, which ensures that the correct substances are measured during the quantitative phase.
In quantitative analysis, two key measurements are made: the sample quantity and a property proportional to the amount of the analyte (the substance being analyzed). This forms the basis of the...
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Gene transcription is regulated by the synergistic action of several proteins that form a complex at a gene regulatory site. This is observed in eukaryotes, where the regulation of gene expression is a complex process. Regulatory proteins in eukaryotes can broadly be classified into two types – regulators that bind directly to specific DNA sequences and co-regulators that associate with regulatory proteins but cannot directly bind to the DNA. These co-regulators are further divided into...
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Aminoacyl-tRNA synthetases are present in both eukaryotes and bacteria. Though eukaryotes have 20 different aminoacyl-tRNA synthetases to couple to 20 amino acids, many bacteria do not have genes for all of these aminoacyl-tRNA synthetases. Despite this, they still use all 20 amino acids to synthesize their proteins. For instance, some bacteria do not have the gene encoding the enzyme that couples glutamine with its partner tRNA. In these organisms, one enzyme adds glutamic acid to all of the...
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Activation Energy01:26

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Activation energy is the minimum amount of energy necessary for a chemical reaction to move forward. The higher the activation energy, the slower the rate of the reaction. However, adding heat to the reaction will increase the rate, since it causes molecules to move faster and increase the likelihood that molecules will collide. The collision and breaking of bonds represents the uphill phase of a reaction and generates the transition state. The transition state is an unstable high-energy state...
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Eukaryotic Transcription Activators02:42

Eukaryotic Transcription Activators

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Transcription activators are proteins that promote the transcription of genes from DNA to RNA. In most cases, these proteins contain two separate domains ‒ a domain that binds to DNA and a domain for activating transcription; however, in some cases, a single domain is responsible for both binding and activation of transcription, as seen in the glucocorticoid receptor and MyoD.
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Secondary Active Transport01:55

Secondary Active Transport

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One example of how cells use the energy contained in electrochemical gradients is demonstrated by glucose transport into cells. The ion vital to this process is sodium (Na+), which is typically present in higher concentrations extracellularly than in the cytosol. Such a concentration difference is due, in part, to the action of an enzyme “pump” embedded in the cellular membrane that actively expels Na+ from a cell. Importantly, as this pump contributes to the high concentration of...
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Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions
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Quantitative analysis of USP activity in vitro.

Shreya Dharadhar1, Robbert Q Kim1, Michael Uckelmann1

  • 1Division of Biochemistry and Oncode Institute, Netherlands Cancer Institute, Amsterdam, The Netherlands.

Methods in Enzymology
|March 10, 2019
PubMed
Summary

This chapter details methods for studying ubiquitin-specific proteases (USPs), crucial deubiquitinating enzymes. It covers construct design, protein purification, and quantitative activity assays for understanding USP function and regulation.

Keywords:
Deubiquitinating enzymesProtein purificationProtein–protein interactionQuantitative enzymatic activity studiesUbiquitinUbiquitin-specific protease

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Area of Science:

  • Biochemistry and Molecular Biology
  • Enzymology
  • Proteostasis

Background:

  • Ubiquitin-specific proteases (USPs) are key deubiquitinating enzymes (DUBs) involved in cellular processes.
  • Understanding USP function and regulation requires precise quantitative activity measurements.
  • Existing methods may not fully capture the complexity of USP activity and regulation.

Purpose of the Study:

  • To provide comprehensive protocols for the quantitative characterization of USP activity.
  • To guide researchers in designing and expressing USP constructs for purification and analysis.
  • To detail methods for studying USP interactions and integrating them into kinetic analyses.

Main Methods:

  • Efficient design and optimal expression of USP constructs.
  • Purification strategies for active USP enzymes.
  • In vitro activity assays using minimal and natural substrates.
  • Binding assays to study protein-protein interactions.
  • Incorporation of regulatory elements and interacting proteins in assays.

Main Results:

  • Established protocols for reproducible purification of functional USP proteins.
  • Demonstrated quantitative kinetic analysis of USP activity on various substrates.
  • Methods for assessing the impact of regulatory domains and interacting partners on USP function.

Conclusions:

  • The presented methodologies enable robust quantitative characterization of USP enzymatic activity.
  • These approaches facilitate a deeper mechanistic understanding of USP function and regulation in cellular physiology.
  • The chapter provides a valuable resource for researchers investigating DUBs and the ubiquitin system.