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.
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.
