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 |
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.