Subcellular fractionation and protein enrichment are important methods in the rapidly growing field of proteomics. Isolation of subcellular fractions and concentration of proteins in low abundance allow for more efficient identification and study of proteins of interest. Examples are the isolation of integral membrane proteins and nuclear proteins.

Membrane protein extraction

Membrane proteins comprise approximately 30% of the eukaryotic proteome and are a key target in drug discovery research. However, they are difficult to isolate because of their hydrophobicity, basic nature and large size.

Certain detergents can be used to selectively extract and isolate membrane (hydrophobic) proteins from cytosolic (hydrohilic) proteins. For example, solutions of Triton X-114 are homogeneous at 0°C (form a clear micellar solution) but separate into aqueous and detergent phases above 20°C (the cloud point) as micellar aggregates form and the solution turns turbid. With increased temperature, phase separation proceeds until two clear phases form. Proteins partition according to their hydrophilic and hydrophobic features. Membrane proteins are enriched in the hydrophobic fraction.

Steps involved in membrane protein extraction.

The Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit enriches for integral membrane proteins and membrane-associated proteins from cultured mammalian cells or tissue via selective solubilization using a mild detergent-based protocol. The use of selective detergent extraction eliminates the hassle of phase separation based on hydrophobicity, allowing better reproducibility and higher throughput. The cells are first permeabilized with a mild detergent, allowing the release of soluble cytosolic proteins, after which a second detergent solubilizes membrane proteins.

Isolation and enrichment of membrane proteins from different tissues. Membrane proteins were isolated from frozen mouse heart or brain (30 mg) following the Mem-PER Plus Membrane Protein Extraction Kit protocol. Membrane and cytosolic fractions (10 μg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Western blots were done using the Thermo Scientific Pierce Fast Western Rabbit Dura Kit (Part No. 35071) and primary antibodies diluted 1:1000. Images were generated using the Thermo Scientific myECL Imager.

Cell and Protein Isolation Technical Handbook

Learn how to optimize protein extraction from cells and tissue for better yield and improved downstream compatibility using Thermo Scientific™ Pierce™ Protein Biology Products.

  • Extract total protein quickly and efficiently
  • Obtain subcellular fractions with minimal cross- contamination
  • Enhance lysis and immunoassay techniques with highly purified, prediluted detergents
  • Remove interfering detergents rapidly and effectively

Nuclear protein extraction

The preparation of good nuclear protein extracts is central to the success of many gene regulation studies. Nuclear extracts are used instead of whole cell lysates for several reasons. First, many experiments in the area of gene regulation are adversely affected by cellular components present in whole cell lysates. Second, the concentration of the nuclear protein of interest is diluted by the vast array of cytoplasmic proteins present in whole cell extracts. Finally, whole cell lysates are complicated by the presence of genomic DNA and mRNA. A variety of methods exist to isolate nuclei and prepare nuclear protein extracts. However, most of these are lengthy processes requiring mechanical homogenization, freeze/thaw cycles, extensive centrifugation or dialysis steps that may compromise the integrity of many fragile nuclear proteins.

Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Reagent kit enables stepwise lysis of cells that generates both functional cytoplasmic and nuclear protein fractions in less than two hours. Cultured mammalian cells or tissues are processed by first disrupting the outer cell membrane to obtain the cytoplasmic contents and then extracting proteins from the nuclei. Cross-contamination between the two fractions is minimal (<10%). With this stepwise fractionation procedure, one can obtain concentrated nuclear extracts without compromising gene regulation experiments, as is commonly seen when whole cell lysates are analyzed. Prepared extracts are compatible with many downstream applications, including electrophoretic mobility shift assays (EMSA) with nuclear extracts, reporter assays with cytosolic extracts, western blots, enzyme assays and the Thermo Scientific Pierce BCA Protein Assay.

Chemiluminescent EMSA of four different DNA–protein complexes. DNA-binding reactions were performed using 20 fmol biotin-labeled DNA duplex (1 Êbiotin per strand) and 2 µL (6.8 µg total protein) NE-PER nuclear extract prepared from HeLa cells. For reactions containing specific competitor DNA, a 200-fold molar excess of unlabeled specific duplex was used.

Subcellular protein fractionation

The Thermo Scientific Subcellular Protein Fractionation Kit enables stepwise separation and extraction of cytoplasmic, membrane, nuclear-soluble, chromatin-bound and cytoskeletal proteins from mammalian cultured cells or tissue. Extracts obtained with the Subcellular Protein Fractionation Kit are compatible with a variety of downstream applications, including western blotting, protein assays, electrophoretic mobility shift assays and reporter-gene and enzyme-activity assays.

Schematic of subcellular fractionation using a commercially available kit. At each step, the supernatant contains the respective subcellular fraction, and the pellet can be used for the subsequent step. The first reagent added to a pellet of cultured cells is buffer A, which causes selective permeabilization of the cell membrane, thereby releasing soluble cytoplasmic contents. The second reagent, or buffer B, dissolves plasma, mitochondria and endoplasmic reticulum-Golgi membranes, but does not solubilize the nuclear membranes. The intact nuclei are then retrieved by centrifugation, and a third nuclear extraction, buffer C, then yields the soluble nuclear extract. Micrococcal nuclease (MNase) can be added to buffer C in an additional step if chromatin-bound nuclear proteins are to be extracted. The recovered insoluble pellet is then extracted with the pellet extraction buffer D, which isolates the cytoskeletal proteins.

Fractionation of subcellular proteins enables protein localization assessment and protein enrichment from specific cellular compartments. The Subcellular Protein Fractionation Kit includes a combination of reagents for stepwise lysis of cells into functional cytoplasmic, membrane, nuclear-soluble, chromatin-bound and cytoskeletal protein fractions in less than 3 hours. Extracts from each subcellular compartment generally have less than 15% contamination between fractions, which is sufficient purity for most experiments studying protein localization and redistribution.

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Nucleus and organelle enrichment

Subcellular fractionation simplifies complex protein mixtures, thereby facilitating proteomic analysis. Isolation of intact organelles enables analysis at either whole organelle or protein-fractional levels. Isolation of organelles is accomplished by cell membrane lysis and density gradient centrifugation to separate organelles from contaminating cellular structures. Intact nuclei and organelles have distinctive sizes in mammalian cells, enabling them to be separated by this method.

We have developed three organelle enrichment kits for lysosomes, peroxisomes and nuclei that enable enrichment of intact organelles from cells and tissue. The isolated organelles may be used for a number of downstream applications, including 2D/MS, electron microscopy, disease profiling, gene expression, signal transduction and interaction or localization studies.

Lysosome, peroxisome and nuclei enrichment. Panel A. Lysosome enrichment from tissue. Liver and kidney tissues (200 mg each) were processed using the Thermo Scientific Lysosome Enrichment Kit for Tissue and Cultured Cells. Total lysate and isolated lysosomes were analyzed by western blotting for Lamp-1, a lysosomal membrane protein marker. Panel B. Peroxisome enrichment from tissue. Liver and kidney tissues (300 mg each) were processed using the Thermo Scientific Peroxisome Enrichment Kit for Tissue. Total lysate and isolated peroxisomes were analyzed by western blotting for PMP70, a peroxisomal membrane protein marker. Panel C. Nuclei enrichment from tissue. Liver and kidney tissue (400 mg each) were processed using the Thermo Scientific Nuclei Enrichment Kit for Tissue. Total cell lysate and isolated nuclei were analyzed by western blotting for histone deacetylase (HDAC), a soluble nuclear marker.

Mitochondria isolation and mitochondrial protein isolation

Isolation of intact mitochondria is typically a laborious process requiring single-sample processing with Dounce homogenization. Density gradient centrifugation approaches are also effective, but these are generally only practical for large-scale needs. When the goal is small-scale enrichment of mitochondria and/or extraction of mitochondrial proteins, a reagent-based microcentrifuge method is desirable.

Dounce homogenizer.

The Thermo Scientific Mitochondria Isolation Kit uses a non-mechanical, reagent-based method that allows multiple samples (up to six) to be processed concurrently. Cultured mammalian cell pellets are gently lysed using a proprietary formulation that results in maximum yield of mitochondria with minimal damage to integrity. Guidelines are given for optimizing purity vs. yield parameters. Also included are instructions for a Dounce homogenization procedure, which results in two-fold more mitochondria recovery compared to the reagent-based method. Both methods use differential centrifugation to separate the mitochondrial and cytosolic fractions with a benchtop microcentrifuge and are completed in approximately 40 minutes (post-cell harvest). Once isolated, the mitochondria can be used in downstream applications such as apoptosis, signal transduction and metabolic studies, as well as to facilitate mitochondrial proteomics efforts.

Recommended reading

  1. Walker JM (2009) The Protein Protocols Handbook. Third Edition. New York (NY): Springer-Verlag New York, LLC.