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Related Concept Videos

Subcellular Fractionation01:32

Subcellular Fractionation

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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...
7.1K

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Updated: Aug 21, 2025

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification
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Subcellular Fractionation for DIGE-Based Proteomics.

Sandra Murphy1

  • 1Charles River Laboratories, Saffron Walden, UK. Sandra.Murphy@crl.com.

Methods in Molecular Biology (Clifton, N.J.)
|November 15, 2022
PubMed
Summary
This summary is machine-generated.

This study details enriching crude microsomes from skeletal muscle using differential centrifugation. This method improves detection of low-abundance proteins for better health and disease insights.

Keywords:
DIGEMicrosomal enrichment strategyMuscle proteomicsSubcellular fractionationTwo-dimensional gel electrophoresisUltracentrifugation

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Area of Science:

  • Analytical Biochemistry
  • Proteomics
  • Cell Biology

Background:

  • Mass spectrometry enables large-scale protein identification but requires effective separation techniques.
  • Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a key separation method.
  • Detecting low-abundance proteins, crucial for understanding health and disease, necessitates optimized fractionation.

Purpose of the Study:

  • To describe experimental steps for enriching crude microsomes from skeletal muscle.
  • To validate enrichment using gel electrophoresis and immunoblotting.
  • To prepare samples for comparative 2D-DIGE analysis.

Main Methods:

  • Differential centrifugation for subcellular fractionation of skeletal muscle.
  • Enrichment of crude microsomes.
  • Verification of enrichment via gel electrophoresis and immunoblotting.

Main Results:

  • Successful enrichment of crude microsomes from skeletal muscle tissue.
  • Verified enrichment through gel electrophoresis and immunoblotting techniques.
  • Demonstrated a viable protocol for subsequent proteomic analysis using 2D-DIGE.

Conclusions:

  • Differential centrifugation is effective for crude microsome enrichment from skeletal muscle.
  • This enrichment protocol enhances the detection of low-abundance proteins.
  • The described methodology supports advanced proteomic studies in skeletal muscle.