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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...
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Subcellular Fractionation for DIGE-Based Proteomics.

Sandra Murphy1

  • 1Department of Biology, Maynooth University, National University of Ireland, Maynooth, Co. Kildare, Ireland. sandra.murphy@nuim.ie.

Methods in Molecular Biology (Clifton, N.J.)
|October 12, 2017
PubMed
Summary
This summary is machine-generated.

This study details enriching crude microsomes from skeletal muscle using differential centrifugation. This method aids in detecting low-abundance proteins crucial for understanding health and disease states via mass spectrometry.

Keywords:
Microsomal 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 sample fractionation.
  • Two-dimensional difference gel electrophoresis (2D-DIGE) is a key separation technique.
  • Low-abundance proteins are often under-represented but critical in biological processes and disease.

Purpose of the Study:

  • To describe a protocol for enriching crude microsomes from skeletal muscle.
  • To validate the enrichment process using gel electrophoresis and immunoblotting.
  • To prepare samples for subsequent comparative 2D-DIGE analysis.

Main Methods:

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

Main Results:

  • Successful enrichment of crude microsomes from skeletal muscle tissue.
  • Demonstrated effectiveness of differential centrifugation for subcellular fractionation.
  • Validated enrichment through gel electrophoresis and immunoblotting.

Conclusions:

  • Differential centrifugation is an effective method for crude microsome enrichment in skeletal muscle.
  • This enrichment is essential for improving the detection of low-abundance proteins in proteomic studies.
  • The described protocol facilitates advanced proteomic analyses like comparative 2D-DIGE.