Cell Fractionation and Organelle Isolation

Cell Fractionation and Organelle Isolation

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Cell Fractionation and Organelle Isolation

What is cell fractionation?

Cell fractionation is a method to separate subcellular components, and isolate organelles and other subcellular components from one another.

What is the purpose of cell fractionation?

Protein Enrichment

Enrich target proteins and improve detection of low abundance proteins.

Protein Characterization

Identify the subcellular localization of a protein.

Protein Translocation

Monitor translocation of cell signaling molecules from the cytoplasm to the nucleus

Fractionation and Extraction Kits


Fractions Isolated

Nuclear/Cytoplasm Kit

Nuclear and cytoplasmic fractions

Mitochondria/Cytoplasm Kit

Mitochondrial and cytoplasmic fractions

Endoplasmic Reticulum Kit

Total ER (rough and smooth) or rough ER alone.

Membrane/Cytoskeletal Kit

Nuclear, cytoplasm, membrane, and cytoskeletal fractions

Antibodies to Confirm Fractionation

It is recommended to confirm the enrichment of subcellular extracts following isolation. Western blot analysis can be used to confirm enrichment by probing a protein whose expression is restricted to the subcellular fraction of interest (e.g. Lamin B1 is expressed in the nucleus, but not the cytoplasm). The nuclear fraction should demonstrate more robust Lamin B1 signal than the cytoplasmic fraction following nuclear/cytoplasmic fractionation.

Antibodies by Organelle/Subcellular Fraction

Nucleus Lamin B1
Histone H2B
Histone H3

Cytoplasm/Cytoskeletal Beta-actin

Endoplasmic Reticulum GRP78

Golgi Apparatus 58K Golgi Protein

Mitochondria Cytochrome c

Lysosome LC3

Membrane N-Cadherin

View All Cellular Marker Antibodies

Tips for Fractionation

Add protease and phosphatase inhibitors to preserve the physiology of your protein:
  • Add protease and phosphatase inhibitors to prevent autolysis and changes in the target protein’s activation state or interactions. Phosphatase inhibitors prevent the removal of phosphate groups from phosphorylated proteins, while protease inhibitors prevent proteases from cleaving peptide bonds and changing protein-protein interactions.

Stimulate or treat your cells with inhibitors to localize your protein.
  • Proteins can shuttle between organelles or be expressed in multiple compartments. Cells may require stimulation or inhibition to restrict the expression of your target protein.