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Detergent Purification of Membrane Proteins01:18

Detergent Purification of Membrane Proteins

Detergents are used to purify the integral proteins of the membrane. The hydrophobic portion of the detergent can replace membrane phospholipids while solubilizing the membrane proteins. When detergent monomers reach a specific concentration in a solution called critical micelle concentration (CMC), they form micelles. Above CMC, the concentration of the detergent monomers remains in equilibrium with the micelle. The number of detergent monomers present in the CMC varies for each detergent, and...

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Activity-Based Protein Profiling of Serine Hydrolases in Bacteria: Methods and Protocols.

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Summary

Activity-based protein profiling (ABPP) enables enzyme activity detection in bacteria using functionalized probes. This study details protocols for fluorescent and biotinylated probes, adaptable for various enzyme families and bacterial types.

Keywords:
Activity-based probeBacteriaChemoproteomicsFlurorophosphonateSerine hydrolases

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Area of Science:

  • Chemoproteomics
  • Enzymology
  • Microbiology

Background:

  • Activity-based protein profiling (ABPP) is a powerful chemoproteomic technique.
  • It utilizes functionalized covalent enzyme inhibitors, known as activity-based probes (ABPs), to interrogate enzyme activity.
  • Understanding active enzymes in bacteria is crucial for various biological and medical applications.

Purpose of the Study:

  • To describe experimental protocols for profiling enzyme activities in bacterial cultures.
  • To detail the use of both fluorescent and biotinylated ABPs for enzyme detection and identification.
  • To provide adaptable methods for targeting diverse enzyme families in bacteria.

Main Methods:

  • Development and optimization of ABPP protocols for bacterial samples.
  • Application of fluorescent ABPs for gel-based detection and resolution of enzyme activities.
  • Utilization of biotinylated ABPs for enrichment and mass spectrometry-based identification of enzymes.
  • Optimization for serine hydrolase profiling using fluorophosphonate probes in Gram-positive and Gram-negative bacteria.

Main Results:

  • Established robust protocols for ABPP in bacterial systems.
  • Demonstrated successful gel-based detection and MS-based identification of active bacterial enzymes.
  • Showcased optimized methods for serine hydrolase profiling.
  • Validated adaptability for other enzyme families and bacterial species.

Conclusions:

  • The described ABPP protocols provide a versatile platform for bacterial enzyme activity profiling.
  • These methods facilitate the detection, quantification, and identification of active enzymes in diverse bacterial contexts.
  • The protocols are readily adaptable for broader applications in bacterial research and drug discovery.