Cell Fractionation and Organelle Isolation

Fractionation and Extraction Kits | Antibodies to Confirm Fractionation | Tips for Fractionation

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

Kit	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. What is the purpose of cell fractionation?
Nuclear/Cytoplasm/ 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 Alpha-tubulin Desmin
Endoplasmic Reticulum	GRP78 Calreticulin Calnexin
Golgi Apparatus	58K Golgi Protein GOLM1 TGN46
Mitochondria	Cytochrome c HSP60 COX IV
Lysosome	LC3 LAMP2 ATG5
Membrane	N-Cadherin CD98 PMCA

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.