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Related Concept Videos

Subcellular Fractionation01:32

Subcellular Fractionation

9.0K
The homogenate obtained after cell lysis contains various membrane-bound organelles that can be further separated into pure fractions by subcellular fractionation. These isolates are used to study specific cellular components, analyze localized protein activity, and are even employed in diagnostics. Fractionation is typically achieved using centrifugation methods, the most common being density-gradient and differential centrifugation.
Differential Centrifugation
Differential centrifugation is...
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Overview Of Cell Separation And Isolation01:20

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Cell separation was first achieved in 1964 by S. H. Seal, who separated large tumor cells from the smaller blood cells using filtration. Two years later, Pohl and Hawk performed experiments on how cells respond differently to a nonuniform electric field based on the cell type. Such observations were the inception of cell separation methods, which allow isolating a single cell type from a heterogeneous sample.
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Centrifugation01:05

Centrifugation

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Centrifugation is a separation technique based on differences in density or size. It is commonly used to separate solids from aqueous interferents. During centrifugation, the sample is placed in centrifugation tubes and spun at high angular velocity, which allows centrifugal force to act differentially on the different densities or masses of the components. After spinning, the supernatant liquid is decanted. Depending on the specific application, either the pellet or the supernatant is retained...
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Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification
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Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

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Cell Fractionation.

Melissa Petiti1, Laetitia Houot1, Denis Duché2

  • 1Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM, UMR 7255), Institut de Microbiologie de la Méditerranée (IMM), Aix-Marseille Université-Centre National de la Recherche Scientifique (CNRS), 31 Chemin Joseph Aiguier, 13402, Marseille Cedex 20, France.

Methods in Molecular Biology (Clifton, N.J.)
|July 2, 2017
PubMed
Summary
This summary is machine-generated.

This study presents a simple fractionation method to isolate cellular compartments in Escherichia coli, aiding in protein localization. The technique helps determine protein presence and differentiate between integral and peripheral inner membrane proteins.

Keywords:
Freeze and thawMembraneOsmotic shockPeptidoglycanProtein solubilisationSpheroplastSubcellular localisation

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • Protein function is intrinsically linked to its subcellular localization within a cell.
  • Gram-negative bacteria like Escherichia coli possess multiple compartments, including the cytoplasm, inner membrane, periplasm, outer membrane, and extracellular medium.
  • Accurate protein localization is crucial for understanding cellular processes.

Purpose of the Study:

  • To describe a simple and efficient method for fractionating Escherichia coli cells.
  • To enable the determination of protein presence within specific cellular compartments.
  • To provide a means to distinguish between integral and peripheral inner membrane proteins.

Main Methods:

  • Cellular fractionation of Escherichia coli.
  • Biochemical techniques for compartment isolation.
  • Analysis of protein presence in isolated fractions.
  • Specific methods to differentiate inner membrane protein types.

Main Results:

  • Successful isolation of distinct cellular compartments from Escherichia coli.
  • Demonstration of the method's efficiency in localizing proteins.
  • A validated approach to classify inner membrane proteins as integral or peripheral.

Conclusions:

  • The described fractionation method is effective for studying protein localization in Escherichia coli.
  • This technique facilitates a deeper understanding of protein distribution and function.
  • The ability to differentiate inner membrane protein types enhances cellular analysis.