Membrane Protein Extraction

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Efficient mammalian membrane protein extraction

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

Evelina Čirbaitė, M.S.1; Scott Meier, M.S.; Juozas Šiurkus, Ph.D.1;

August 29, 2013


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 which 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, 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.

Figure 1. Protocol summary for Mem-PER Plus Kit

Figure 1. Schematic presentation of the membrane protein extraction work flow with Mem-PER Plus Membrane Protein Extraction Kit from the mammalian cells. First, the cells are permeabilized with a mild detergent containing the Permeabilization Buffer, with the aim to liberate soluble cytosolic proteins. After removal of the soluble fraction containing hydrophilic proteins, the membrane proteins are extracted from the insoluble fraction using the Membrane Solubilization Buffer. Total time required:  ≤ 90 minutes.


RESULTS and DISCUSSION:

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 4oC.

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).

Figure 2. Comparison of membrane protein extraction kits

Figure 2. Improved protein yield using Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit. Membrane proteins were isolated from mouse liver tissue, Jurkat cells and HeLa cells using four commercial extraction kits (see METHODS section for details). Protein yields (micrograms) for membrane, cytosolic and total fractions were determined with the Thermo Scientific Pierce BCA Protein Assay Kit (Part No. 23225).

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.

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.

Figure 4. Efficient extraction of multiple membrane spanning proteins in various cell lines.

Figure 4. Efficient extraction of multiple membrane spanning proteins in various cell lines. Membrane proteins were isolated from 5 x 10^6 cultured cells following the Mem-PER Plus Membrane Protein Extraction Kit protocol. Membrane fractions (30mg) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies against the corresponding protein and HRP-tagged secondary antibody. Blots were developed with Super Signal West Dura Substrate and 1-minute exposures in the myECL Imager.


CONCLUSIONS:

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.


METHODS:

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.


GENERAL REFERENCES:

  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.

12 comments to Efficient mammalian membrane protein extraction

  • Moo

    Hi,

    I’m just wondering whether you can get a chance to test human monocytic cell lines such as THP1 cell lines to isolate membrane proteins using this kit?

    Thanks

    Moo

  • Carlo van Roermund

    Hi,
    I am trying to extract membrane and cytosol fractions from yeast peroxisomes. What kit is the best kit to use for isolating the peroxisomal membrane protein. After that I will measure the transport activity of these proteins in liposomes. So I need after the extraction an active membraneprotein. Any suggestion what kit I can use to isolate membrane proteins?

    Thanks,

    Carlo

  • Tushar Deshpande

    Hi,
    I would like to know about the term “membrane protein” used here. Does the kit separates all membrane proteins (mitochondrial, ER, plasma membrane) or it selectively separates plasma membrane proteins from all cytosolic proteins (which is mixture of true cytosolic proteins and cytosolic organnels like Endoplasmic reticulum-ER, mitochondria)?
    Does the membrane fraction obtained by this kit also contain proteins from ER or golgi? This is very important since the protein of my interest is present at plasma membrane as well as in the cytoplasmic organells like ER, golgi etc. It will be of great help if you mention atleast the principle (and not the detailed mechanism) with which the kit works!

    Thank you in appreciation,

    • Scott

      Hello Tushar,

      This kit works on the principle of selective solubilization. Cells are permeabilized to release non-membrane enclosed soluble cytosolic proteins, and then pelleted. The pellet containing all membranes and membrane enclosed organelles are then solubilized with another detergent formula that releases integral membrane proteins, peripheral membrane proteins, as well as proteins in membrane enclosed organelles.

      The “membrane” fraction will indeed include plasma as well as mitochondrial, ER, Golgi, and even nuclear membrane proteins. This kit is not for the selective isolation of plasma membrane proteins. If you are interested in isolating only cell surface protein, you may want to try our cell surface protein isolation kit (http://www.piercenet.com/product/pierce-cell-surface-protein-isolation-kit).

  • Nicole

    Can you isolate membrane proteins from Pichia Pastoris with this kit?

    • Scott

      Hello Nicole,

      We have not tested isolation of membrane proteins using Mem-PER Plus on yeast cells. It is not known at this time whether or not the permeabilization buffer is able to poke holes in the cell wall of yeast. Original Mem-PER (Product #89826) has been tested on membrane protein isolation from S. cerevisiae, with disruption of the cell wall using glass beads.

      We have sample sizes available for the Mem-PER Plus reagent that you could request if you wanted to evaluate compatibility with Pichia.

  • Sam

    Hi,
    I am trying to extract membrane and cytosol fractions from human macrophages (7.5 million cells) and detect caveolin-1 via western blot. However, protein concentration yield seems to be very low for cytosol fraction (~4ug). Any suggestion as to how I can increase the yield?

    Thanks,
    Sam

    • Scott

      Hello Sam,

      While we have tested several cell lines and tissues for fractionation of membrane and cytosolic proteins using the Mem-PER Plus Kit, sadly macrophages were not one of them. First, confirm that cytosolic proteins are not contaminating the membrane fraction probing for MEK1/2 by western blot. If there is cytosolic contamination of the membrane fraction, longer incubation times (up to 30 minutes) with gentle agitation may increase the permeabilization of the cell membrane and release of soluble cytosolic protein into the cytosolic fraction. Also, always include protease inhibitors in the cell permeabilization and membrane solubilization buffers to avoid protein degredation. If you are using frozen instead of fresh cells, wash the cells before freezing. Thaw the cells before use, and proceed to the cell permeabilization step. Freeze/thaws can rupture the cells, releasing the cytosolic protein into the washes instead of the cytosolic fraction.

  • Andreia

    I would like to know if this kit is mass spec compatible, for further analysis.

    Many thanks in advance.

    • Scott

      Hi Andreia,

      The membrane permeabilization and solubilization buffers included in this kit contain detergents that are not directly mass spec compatible. Sample cleanup is necessary for downstream analysis by MS, with your best bet being SDS-PAGE and in-gel digestion. It may be possible to remove the detergents by dialysis or acetone precipitation, however we have not evaluated this.

  • Subhasree Basu

    HI,
    I am a post doc fellow in Wistar Institute. I am wondering if I can please get a sample of the kit to try plasma membrane extraction. Kindly let me know.
    Thanks
    Subhasree

    • Kevin

      Hi, Subhasree. Most of our products do not have sample sizes available, especially multi-component kits. However, I’m going to pass your request along to the product manager for that product and have her contact you.

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