Cytoskeletal enrichment assays (right) enhance results for immunodetection methods including IHC and WB
Although the study of cells derived from in vivo tissues is most predictive of cellular biology and function, biological complexity can confound results unless target cell populations are isolated. In order to study cell phenotypes, diverse methods have evolved for isolating cell populations based on definitive characteristics. Isolating cell populations not only aids biological research, but also facilitates clinical diagnostic testing. The most effective isolation methods demonstrate enhanced yield, purity, and viability. Enrichment or purification of target populations can be achieved by positive selection─often employing antibodies that bind cell-specific markers─or by negative selection that may use biophysical properties to deplete unwanted cells in order to enrich for the target population.
Fractionation of cells into their subcellular components has long been fundamental to cell biology studies. Subcellular fractionation techniques have been widely used to study structure and function of organelles and subcellular compartments, as well as to understand the location, processing, and trafficking of biomolecules. The goal of most fractionation techniques is to obtain organelles and cellular macromolecules in a functional state, where they retain their intrinsic biochemical properties. This is often achieved by cell lysis employing gentle mechanical means or with mild detergents, frequently followed by fractionation of cellular components by differential centrifugation.
Preadipocytes can be used to study the process of adipogenesis and, post-differentiation, to assess lipolysis, cell signaling, endocrine function, and metabolic activity.
Our Preadipocyte Isolation Kit combines the tools and reagents needed to isolate the stromal vascular cell fraction from adipose tissue. The resulting stromal vascular fraction is cultured on a tissue-culture plate, where preadipocytes adhere. The preadipocytes retain the ability to proliferate and differentiate into adipocytes when treated with differentiation-inducing components.
Visible lipid droplets confirm the isolation of preadipocytes 7 days after differentiation using 3T3-L1 Differentiation Kit.
Derivation of subcellular components promotes understanding of organelle function and can lead to new biotechnologies. For example, nuclei isolated from mammalian cells can subsequently be used for the synthesis of endogenous RNA primary transcripts. The high-yield Nuclei EZ Prep Kit (NUC101, NUC201) was developed for the rapid isolation of nuclei from most mammalian cells.
For the detection of mitochondrial integrity and membrane potential, our mitochondrial staining kit (CS0390, CS0760) uses the JC-1 dye (420200, T4069), which concentrates in the mitochondrial matrix to form red fluorescent aggregates. Events like apoptosis that dissipate the mitochondrial membrane potential prevent the accumulation of JC-1 dye in the mitochondria, and a fluorescence microscope can then be used to distinguish viable from compromised mitochondria.
Non-organelle subcellular complexes like proteasomes can also be isolated for use in proteolytic assays. Our method uses affinity matrix beads containing a GST-fusion protein with a ubiquitin-like domain bound to GST-agarose.
Working with the proteins that associate with the actin cytoskeleton has traditionally been a challenge due to the insolubility of cytoskeletal elements in detergents like Triton X-100. The Millipore ProteoExtract® Cytoskeleton Enrichment and Isolation Kit supplies cytoskeleton purification buffers that retain focal adhesion and actin-associated proteins while removing soluble cytoplasmic and nuclear proteins from the cell. This approach enhances the ability to detect and study low-abundance actin-associated proteins which are often masked in Western blotting methods for analysis of whole cell lysates.
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