Differential detergent fractionation of isolated hepatocytes: Biochemical, immunochemical and two‐dimensional gel electrophoresis characterization of cytoskeletal and noncytoskeletal compartments

Abstract

Two-dimensional (2-D) gel electrophoresis is often used in toxicologic and metabolic studies to assess treatment- or stage-specific changes in protein synthesis, degradation or posttranslational modification. When combined with cell fractionation studies the detectability of low abundance proteins is enhanced, and changes in subcellular distribution of proteins can also be monitored. Detergent fractionation is a simpler alternative to differential pelleting, which partitions cellular constituents into functionally distinct populations while preserving cytoskeletal integrity. We defined and characterized a differential detergent fractionation (DDF) protocol to enable protein dynamics in cytoskeletal and noncytoskeletal compartments of isolated hepatocytes to be monitored simultaneously. Rat hepatocytes were maintained in suspension culture and fractionated by sequential extraction with detergent-containing buffers (digitonin/EDTA, Triton/EDTA, Tween/deoxycholate). DDF reproducibly yielded four electrophoretically distinct fractions enriched in cytosolic, membrane-organelle, nuclear membrane and cytoskeletal-matrix markers, respectively. Immunoblotting with over 20 different antibodies corroborated the selectivity of fractionation and was used to characterize the distribution profiles of cytoskeletal (actin, tubulins, cytokeratins, vinculin, myosin, desmoplakins, fodrin, nuclear lamins) and noncytoskeletal proteins (heat-shock 70 proteins, glutathione-S-transferase, calpains, carbamoyl phosphate synthetase, etc.), as well as to identify spots in 2-D gels. Detergent buffers were compatible with equilibrium or nonequilibrium 2-D gel electrophoretic analysis. Extensive 2-D maps of acidic and basic proteins in each fraction were generated along with a tabular listing of M(r) and pI. Thus, DDF reproducibly partitions hepatocytic proteins into functionally distinct cytoskeletal and noncytoskeletal compartments that are readily analyzed by 2-D gel electrophoresis. DDF is simple, applicable to use with other cell types or culture systems and is especially useful when biomaterial is limited (i.e., clinical studies).

MUC1 regulates nuclear localization and function of the epidermal growth factor receptor

“…Cells were then incubated at 37°C for 120 minutes to induce receptor endocytosis and trafficking. Cells were then fractionated by differential detergent fractionation, which takes advantage of the ionic properties of different detergents to isolate the membrane (Triton X-100), cytosolic (digitonin) and nuclear (Tween40) protein fractions (Ramsby et al, 1994). Note that the membrane fractions include plasma membrane, mitochondria, Golgi, endosomes and the endoplasmic reticulum (ER).…”

“…Proteins isolated from the membrane, cytosolic and nuclear compartments were then separated by SDS-PAGE and examined by subsequent immunoblotting to determine the effects of MUC1 expression on EGFR localization. To confirm fraction purity and equal loading, expression of proteins known to localize in each cellular compartment were analyzed: plasma membrane, IGF-1R; ER membrane, BAP31; Endosomes, EEA1; cytosol, myosin IIa; nucleus, histone H3 (Ramsby et al, 1994).…”

“…The differential detergent-fractionation protocol followed was modified from a published method (Ramsby et al, 1994). Before lysis, cells were washed, collected and resuspended in cold PBS.…”

Polarized P-glycoprotein expression by the immortalised human brain endothelial cell line, hCMEC/D3, restricts apical-to-basolateral permeability to rhodamine 123

“…hCMEC/D3 whole cell lysates were separated into cytosolic, membrane/organelle, nuclear and cytoskeletal fractions using differential detergent fractionation as described by Ramsby et al (Ramsby et al, 1994). Briefly, hCMEC/D3 cells were grown to confluence in 75 cm …”

The circadian protein Clock localizes to the sarcomeric Z-disk and is a sensor of myofilament cross-bridge activity in cardiac myocytes

“…This method uses a detergent-based protocol [10] and has been previously described [11]. Cellular proteins were sequentially extracted into four compartments: cytosolic, membrane/organelles, nuclei and cytoskeleton.…”