Novel Technology Transforms Cell Fractionation
Cell fractionation is the gateway to understanding the properties and functions of numerous cellular components. Fractionation involves disrupting the cell mechanically or chemically, followed by centrifuging the lysate to isolate fractions populated by organelles and cell membrane fragments by size and density. Subsequent analysis reveals the numbers, physiologic state, and contents of various cellular components and structures for proteomics, or studies in protein trafcking, signal transduction, and protein-protein/protein-nucleic interactions. Traditionally, lysis is accomplished through grinding, mechanical blending, freeze-thawing, sonication, or through the efects of detergents.
The signifcance of cell fractionation, particularly for the eventual isolation of membrane proteins and constituents, cannot be understated. More than 70% of drug targets and biomarkers are found on plasma membranes, making isolation of membrane proteins one of
the most common protocols in biomedical research.
When all goes well, cell-fractionation products reveal a complete picture of the state of the cell at the time of fractionation, which may be compared through parallel analysis under diferent conditions. For example protein-trafcking studies can provide insights into disease progression or treatment efcacy by comparing “before” and “after” organelle protein contents.
Challenges with conventional methods
How cell disruption occurs is critical for minimizing cross-contamination of organelle components. Conventional cell fractionation involving mechanical disruption and density ultracentrifugation is unpredictable and highly variable, where most of the inconsistency derives from the homogenization step. Traditional homogenizers or blenders produce inconsistent lysates due to the many variables involved in homogenization, for example cell density, bufer conditions, temperature, time—even the mechanical force and cycle number associated with homogenization. No a priori procedural guidelines or protocols defne every situation, or even conditions a typical biologist is likely to encounter.
Cell-fractionation methods lumbered on for decades until 2012, when Invent Biotechnologies released its patented spin-column-based platform, under the trademark Minute™, for rapid cell fractionation that did not require homogenization or ultracentrifugation. By eliminating uncontrolled variables associated with solution-based fractionation methods, Minute™ transforms tedious, unpredictable mechanical cell fractionation with a method involving just a few spins of a tabletop centrifuge.
Minute™ spin column prep maintains the structural integrity of nuclei and other organelles, resulting in clear isolation of cellular components, thus providing new insights into protein trafcking, localization, or translocation.
Spin columns have been in use for decades in the life sciences. A spin column is a solid-phase extraction or fltration tube adapted to work with centrifuges rather than through gravity. In a typical Minute™ protocol, cells are frst sensitized with a proprietary bufer, transferred to a flter cartridge, and centrifuged through the 60 micron pore-size polymeric flter medium. Cells rupture as they zig-zag through the pores inside the flter.
By controlling the variables of cell disruption, Minute™ provides consistent results every time, in part by eliminating the uncertainties associated with traditional homogenization and gradient density centrifugation. Guesswork, and cellular contamination, are eliminated or greatly reduced. Native plasma membrane vesicles and other organelles are further separated from cell lysate by subsequent diferential and density centrifugation with only a regular tabletop microcentrifuge.
Invent Biotechnologies ofers a next-generation platform for quickly isolating plasma membrane and almost any organelle with biological signifcance, all of which are in true native state, without use of any detergents or harmful chemicals.
Spin-column-based cell-fractionation kits are valuable tools for following the movement and interactions of the proteins of interest. In a recent study on the role of the exchange protein EPAC1 during the uptake of Ebola virus into vascular endothelial cells, investigators at the University of Texas and Chinese collaborators infected and washed human umbilical vein epithelial cells with phosphate bufered saline, followed by utilization of the Minute™ Endosome Isolation and Cell Fractionation Kit. Subsequent protein and RNA analysis demonstrated a previously unknown role for EPAC1 in Ebola virus internalization via regulation of phosphatidylinositol-4,5-bisphosphate 3-kinase pathway-mediated floviral macropinocytosis.
In a key experiment to identify diferent forms of Gag-RNA adducts present in diferent subcellular locations. Kutluay et al. studied the changes in Gag-RNA binding to viral RNA during HIV-1 virion assembly. 4SU-fed 293T cells were transfected with HIV-1 proviral plasmid and fractionated by the Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit. The results indicate that the matrix domain of Gag binds exclusively to specifc tRNAs in the cytosol, and this association regulates Gag binding to plasma membrane. This conclusion can’t be drawn without clear separation of cytosolic and plasma membrane fractions.
In another recent study conducted by Xu et al., the Minute™ Plasma Membrane Protein Isolation and the Cell Fractionation Kit and Minute™ Endosome Isolation as well as the Cell Fractionation Kit were used to study the trafcking of IFN-rR2 protein and it was found that infammatory macrophages from E-selectin-defcient mice have less surface IFN-rR2 and impaired IFN-r signaling. This fnding elucidates the mechanism of cellular signaling driven by IFN-rR2 membrane translocation.
The Minute™ spin-column-based platform for cell fractionation provides clear advantages in speed, ease of use, reliability, and consistency over conventional methods. Minute™ ofers a complete collection of fractionation kits including products for plasma membrane, native intact nuclei, mitochondria, endosomes, lysosomes, Golgi apparatus, endoplasma reticulum, lipid rafts, nuclear envelope, nuclear matrix, proteasomes, chloroplasts, and plant microsomal membrane. Try out one of our kits for your next cell-fractionation study, and see what all the excitement over spin-column fractionation technology is all about!