Efficient Mammalian Membrane Protein Extraction

Fast and simple enrichment of integral membrane proteins and membrane-associated proteins

byEvelina Čirbaitė, M.S.1; Scott Meier, M.S.; Juozas Šiurkus, Ph.D.1 - 08/29/13

Integral and peripheral membrane proteins (MPs) are important for the maintenance of many cellular functions such as signal transduction, cell integrity, intracellular and extracellular transport of molecular solutes and cell-to-cell communication. The physical nature of the association between integral or peripheral MPs and the membrane  phospholipid bilayer is significantly different. Integral MPs span the entire phospholipid bilayer with one or more segments composed of hydrophobic residues that interact with the fatty acyl groups of the membrane phospholipids. In addition, some integral MPs are monotopic, and are embedded in only one leaflet of the bilayer.

In contrast, peripheral MPs do not span the phospholipid bilayer. Non-integral MPs are usually transiently immobilized on the surface of the cytoplasmic face of the plasma membrane via interaction either with polar groups of the lipids and/or surface-spanned integral MPs, or can be anchored into the membrane through post-translational lipidation.

Membrane proteins and receptors are the largest category of druggable targets studied in the pharmaceutical industry. Approximately two-thirds of currently available therapeutic molecules target one or more MPs. Where available, membrane protein crystal structures facilitate elucidation of their functions and discovery of mechanisms of MP-drug molecule interactions. However, despite the high abundance of MPs in the cell proteomes (20-30% of all cellular proteins), only 0.1% of MPs crystal structures have been determined to date.

Methods to characterize MPs are limited by the lack of extraction protocols and reagents that allow sufficient amounts of MPs to be obtained from various cell types without cross-contamination from other protein fractions. There are many strategies to extract MPs from eukaryotic cell lines and tissues, including subcellular fractionation (e.g., sucrose or sorbitol density ultracentrifugation), cationic colloidal silica absorption, aqueous-polymer two-phase system, Thermo Scientific Triton X-114 phase separation, and high-salt or high-pH buffers. Most of these traditional protocols are laborious, time-consuming and require expensive ultracentrifugation equipment.

The Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit enables small-scale solubilization and enrichment of integral and membrane-associated proteins from cultured cells and tissues using a simple reagent-based procedure and a bench-top microcentrifuge (Figure 1). The Mem-PER Plus Kit (Part No. 89842) replaces our former Mem-PER Kit (Part No. 89826), which was based on phase separation with Triton X-114. In this article, we present data to demonstrate the performance of the newer Mem-PER Plus Kit.

With the earlier-generation Mem-PER Kit, extraction resulted in a viscous detergent-based hydrophobic fraction containing MPs, which required further processing before downstream procedures such as protein concentration determination, SDS-PAGE, and western blotting. The procedure for the newly developed Mem-PER Plus Kit requires no phase-separation step; thus the extraction of MPs is simple, highly reproducible, and compatible with downstream applications. Also, the new Mem-PER Plus Kit is suitable for extraction of temperature-sensitive MPs with the entire extraction procedure conducted at 4°C.

We evaluated membrane protein extraction efficiencies from various mammalian cell lines and several types of mouse tissues using various commercially-available extraction kits: Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit, Thermo Scientific Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (the first generation kit), EMD Millipore ProteoExtract Native Membrane Protein Extraction Kit (Merck), and Bio-Rad ReadyPrep Protein Extraction Kit Membrane I (Bio-Rad). We used these kits to extract membrane proteins from a tissue sample and two strains of cultured cells (see METHODS section for details). We compared overall yields of the resulting cytoplasmic and membrane protein fractions (Figure 2).

Protein quantitation of the cytoplasmic and membrane protein fractions demonstrated that the highest yield of MPs was obtained using the Thermo Scientific Mem-PER Plus Kit (Figure 2). A comparable yield was obtained with the ProteoExtract Native Membrane Protein Extraction Kit.

The purity (low cross-contamination) of membrane and cytosolic protein fractions is as important as yield. To verify that the high yields obtained with the Mem-PER Plus Kit were also high-quality extractions of membrane proteins, we performed western blot analysis of membrane and cytoplasmic fractions (Figure 3) obtained from cell and tissue samples. Probing for common membrane protein markers showed that the membrane proteins COX-IV and pan-Cadherin were highly enriched in the membrane fraction, with very low cross-contamination into the cytosolic fraction. (Jurkat cells are non-adherent and do not express cadherins, thus no cadherin was detected in that membrane fraction.) In addition, less than 10% of the cytoplasmic protein Hsp90 was detected in the membrane protein fraction extracted from tissue or cells.

Figure 3. Efficient enrichment of membrane proteins from tissues and cell lines. Membrane proteins were isolated from 30mg of tissue (mouse brain and heart) or 5 x 10^6 cultured cells (Jurkat, HeLa or HEK-293 cell lines) using the Mem-PER Plus Membrane Protein Extraction Kit protocol. Membrane and cytosolic fractions (10mg) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and evaluated by chemiluminescent Western blot for pan-Cadherin, COX-IV, and Hsp90. (Tissue blots were imaged using the myECL Imager; cultured cell blots were imaged by exposure to X-ray film.)

The efficiency of extraction of MPs having multiple membrane spanning domains is also an important concern with MP-extraction protocols. To evaluate the Mem-PER Plus Kit in this regard, we probed for multiple membrane spanning proteins in Western blots of extracts obtained from several cells lines using the Mem-PER Plus Membrane Protein Extraction Kit (Figure 4). The kit effectively extracted membrane proteins ranging from 1 intra-membrane domain (Caveolin-1) to 10 transmembrane domains (Na+/K+ ATPase alpha subunit). HepG2 and LnCAP cells do not express endogenous caveolins. Protease activated receptor-2 (PAR-2), a G-protein coupled receptor, was detected in the membrane fraction of HepG2 cells only. These results suggest that extraction efficiency is dependent upon the expression level of the particular MP and the fluidity of the membrane microenvironment (e.g. lipid rafts rich in cholesterols) in particular cells.

The new Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit was developed for the enrichment of integral and membrane-associated proteins from cultured mammalian cells or tissues. Isolation of MPs is based on a mild detergent selective extraction protocol. The new protocol is easier to perform than the first-generation Mem-PER Kit protocol and results in minimal contamination of cytosolic proteins. After extraction with the Mem-PER Plus Kit, the isolated membrane protein fraction can be used directly in many downstream applications, including protein estimation, SDS-PAGE, and Western blotting.

Membrane protein extraction

Membrane proteins were isolated using the following products, each according to its protocol:

• Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit (Part No. 89842)
• Thermo Scientific Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Part No. 89826)
• EMD Millipore ProteoExtract™ Native Membrane Protein Extraction Kit (No. 444810, Merck)
• Bio-Rad ReadyPrep Protein Extraction Kit (Membrane I) (No. 163-2088, Bio-Rad)

Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit (Part No. 89842) protocol:

• Mouse tissue: Mouse tissue (heart, brain, liver) was excised, weighed and rinsed in phosphate-buffered saline (PBS). The remaining steps were performed according to the instructions for the Mem-PER Plus Membrane Protein Extraction Kit for Soft Tissue (Part No. 89842). The tissue was first washed in Cell Wash Solution, and homogenized in the Permeabilization Buffer using scissors and Dounce tissue grinder. The homogenate was transferred to a new 2mL tube and incubated at 4°C for 10 minutes with constant mixing. Cytosolic proteins were separated by centrifugation at 16,000 x g for 15 minutes at 4°C. The pellet was resuspended in Solubilization Buffer and incubated at 4°C for 30 minutes with constant mixing. Membrane and membrane-associated proteins were recovered by centrifugation of 16,000 x g for 15 minutes at 4°C.
• Mammalian cells: Adherent mammalian cells were harvested using a cell scraper, and approx. 5 x 10^6 cells were resuspended in the growth media. The remaining steps for both adherent and suspension cell lines were performed according to the instructions for the Mem-PER Plus Membrane Protein Extraction Kit for Adherent/Suspension Mammalian Cells (Part No. 89842). Harvested cell suspension (approx. 5 x 10^6 cells) was centrifuged at 300 x g for 5 minutes. The cell pellet was washed twice with Cell Wash Solution and centrifuged at 300 x g for 5 minutes. The cells were resuspended in Permeabilization Buffer and incubated at 4°C for 10 minutes with constant mixing. Cytosolic proteins were separated by centrifugation at 16,000 x g for 15 minutes at 4°C. The pellet was resuspended in Solubilization Buffer and incubated at 4°C for 30 minutes with constant mixing. Membrane and membrane-associated proteins were recovered by centrifugation of 16,000 x g for 15 minutes at 4°C.

Determination of the protein concentration

Protein concentration and yield was determined using the Pierce BCA Protein Assay Kit (Part No. 23225) and Bovine Serum Albumin Standards (Part No. 23208), according to the Microplate Procedure protocol. All protein samples were evaluated directly after extraction, except that membrane protein fractions from the Mem-PER Kit (Part No. 89826) and the Bio-Rad ReadyPrep™ Kit (Product 163-2088, Bio-Rad) were first dialyzed overnight at 4°C against the dialysis buffer (25mM HEPES, 10mM NaCl, 0.5% CHAPS).

Western blot analysis

Equal amounts (based on protein assay) of total protein (10µg or 30µg) were resolved on denaturing 4-20% Tris-glycine SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA in Tris-buffered saline with Tween™ 20 for 1 hour. Membranes were then probed with the appropriate primary antibodies at 1:1000 dilution for 1 hour at room temperature, followed by the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody diluted 1:20,000 (Part No. 31430) or (Part No. 31460). Bands were detected using Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080). Images were generated using the Thermo Scientific myECL Imager (Part No. 62236) or using X-ray film.

Primary antibodies

The following Thermo Scientific Primary Antibodies were used:

• EGFR (epidermal growth factor receptor) (Ab No. PA1-1110)
• COX-IV (cytochrome c oxidase 4) (Ab No. PA5-21359)
• Pan-Cadherin (Ab No. PA5-17526)
• Hsp90 (heat-shock protein 90) (Ab No.MA5-14866)
• Cavelolin-1 (Ab No. PA1-064)
• Na+/K+ ATPase alpha subunit (Ab No. PA5-17251)

The following antibody was from Cell Signal Technology:

• PAR-2 (protease activated receptor-2 ) (No. 6976S)

Cell cultures and mice

Jurkat cells (human T cell leukemia) and LnCAP cells (androgen sensitive human prostate adenocarcinoma) were cultured at 37°C with 5% CO2 in RPMI-1640 with 10% fetal bovine serum with 1% L-glutamine/gentamicin. HeLa (human cervix epitheloid carcinoma), A431 (human epidermoid carcinoma), A549 (human alveolar basal epithelial adenocarcinoma), and HEK293 (human embryo kidney cells) cells were cultured at 37°C with 5% CO2 in DMEM with 10% fetal bovine serum with 1% L-glutamine/gentamicin. Experimental mice (Balb/c, 6-8 weeks old, female) from Vilnius University Institute of Biochemistry animal facility were used to obtain mouse tissue. Experiments were performed as approved by the State Food and Veterinary service of Lithuania, and conducted in accordance with the Law on the Care, Welfare and Use of Animals by the Ethics Commission on the Use of Laboratory Animals, at the State Food and Veterinary Service of Lithuania.

1. Speers, A. E. and Wu, C. C. (2007). Proteomics of integral membrane proteins – theory and application. Chem. Rev. 107: 3687-714. 
2. Arinaminpathy, Y., et al. (2009). Computational analysis of membrane proteins: the largest class of drug targets. Drug Discov. Today. 14(23-24): 1130-5.