The Mem-PER Mammalian Membrane Protein Extraction is an excellent alternative to gradient methods or ultracentrifugation.

• Minimal cross-contamination (typically <10%) of hydrophilic protein into the hydrophobic (membrane) protein fraction (Figures 1 and 2)
• Procedure suitable for a variety of cultured mammalian and yeast cells and mammalian tissues (Figures 1-3)
• Requires only a benchtop microcentrifuge and tubes
• Compatible with downstream applications including SDS-PAGE, Western blotting, protein assays, etc. (Slide-A-Lyzer MINI Dialysis Units and the PAGEprep Kit can aid in these procedures)
• Includes an easy and complete protocol for isolation of membrane proteins in approximately 1 hour from mammalian cells and tissue as well as yeast cells

Traditional methods for isolation of membrane protein are tedious, timeconsuming, and require gradient separation and expensive ultracentrifugation equipment. The faster, easier and less expensive way to isolate membrane proteins is Mem-PER Eukaryotic Membrane Protein Extraction Kit. The three-reagent kit enables extraction and selective enrichment of integral and attached membrane proteins from cultured mammalian and yeast cells, as well as from hard and soft mammalian tissues.

Preparation of mammalian membrane protein extracts using Mem-PER Mammalian Membrane Protein Extraction Kit can be done quickly and efficiently as illustrated below. First, cells are lysed with a detergent and then a second detergent is added to solubilize the membrane proteins. After a quick centrifugation, the cocktail is incubated at 37°C to separate the hydrophobic proteins from the hydrophilic proteins through phase partitioning.

Mem-PER Reagent Protocol

The Mem-PER Kit was found to be highly efficient in the extraction of integral membrane proteins containing one or two transmembrane spanning domains. These results were found to be consistent among three different cell lines tested: NIH 3T3, HeLa and C6. As shown in Figure 2 and Table 1, the integral membrane protein flotillin, containing two transmembrane domains, was extracted with an efficiency of approximately 50% from the three cell lines. These reported values were found to be reproducible in several independent experiments. Extraction of cytochrome oxidase subunit 4 (Cox4), containing one transmembrane domain, was found to be even more efficient with approximately a 90% recovery in the hydrophobic fractions obtained from the three cell lines. Cross-contamination of cytosolic and peripheral proteins into the prepared hydrophobic fraction was minimal (<10%).

Acetylcholinesterase (AchE), a peripheral protein, and Heat shock protein 90 (Hsp90), a cytosolic protein, were found to partition into the hydrophilic fraction with a typical efficiency of >90%. The remaining 10% or less found in the hydrophobic fraction is likely caused in part by difficulty in obtaining complete separation at the interface between the two phases, and the slow disappearance of the interface over time at room temperature. Extraction efficiencies will vary depending on the number of times the integral membrane protein(s) of interest spans the lipid bilayer. Membrane proteins with up to four transmembrane domains are typically extracted with an efficiency of up to 90%. Cross-contamination of cytosolic proteins into the membrane fraction is typically < 10%.

The Mem-PER Kit is compatible with many downstream applications. The isolated membrane protein fraction can be used directly in SDS-PAGE and Western blotting (Figure 3). Protein assays, subsequent purification, etc. can be performed following removal of Reagent C through dialysis using convenient Pierce Slide-A-Lyzer MINI Dialysis Units or Dialysis Cassettes. To effectively remove Reagent C and simultaneously maintain protein solubility, perform dialysis overnight at 4°C with a buffer that includes 0.5% detergent . Quantification of extracted membrane proteins with the Pierce Micro BCA Protein Assay Reagent Kit (Product # 23235 ) results in approximately 250 µg of total protein from 5 x 10⁶ C6 cells. The total amount of protein obtained will vary depending on the cell line. The PAGEprep Clean-Up and Protein Enrichment Kit (Product # 89888) is another Pierce tool for detergent removal. As the name suggests, the PAGEprep Kit prepares samples for SDS-PAGE analysis through the removal of gel-incompatible material. Reagent C in the Mem-PER Kit causes band distortion upon electrophoresis of low molecular weight proteins. The latter can be remedied through treating Mem-PER Reagent-isolated membrane fractions with the PAGEprep Kit. The membrane fraction probed for Cox4 (17 kD) in Figure 2 was treated with the PAGEprep Kit prior to electrophoresis. Electrophoresis of excessive amounts of Reagent C can cause lane distortion; therefore, PAGEprep Reagent is also recommended for analysis of proteins in low abundance where a large volume must be run on SDS-PAGE for adequate detection.

Figure 1. Partitioning of solubilized yeast proteins using the Mem-PER Kit. This experiment was run in triplicate. Yeast proteins from S. cerevisiae (Strain: EGY-194) were solubilized and partitioned based on hydrophobic phase separation. Several proteins corresponding to membrane-bound plasma membrane, organellar membrane and soluble cytosolic proteins from the yeast S. cerevisiae were solubilized and extracted using the Mem-PER Kit. Partitioning efficiency was determined through Western blot analysis of the different proteins. A negligible amount of protein was found in all debris fractions (data not shown). Abbreviations: mitochondrial porin (MP), 3-phosphoglycerate kinase (PGK), alkaline phosphatase (AP), Dol-O-Man synthase (Dpm1p), solubilized membrane protein (M) and hydrophilic protein fraction (H).

Figure 2. Partitioning of solubilized mammalian proteins using the Mem-PER Kit. Proteins from three cell lines were solubilized and extracted using the Mem-PER Kit. Hydrophilic and hydrophobic (membrane protein) fractions were analyzed by Western blot with HRP-labeled secondary antibody followed by SuperSignal West Femto Chemiluminescent Substrate for four proteins from the cellular locations noted. A negligible amount of protein was found in all debris fractions (data not shown). Abbreviations: acetylcholinesterase (AchE), cytochrome oxidase subunit 4 (Cox4), heat shock protein 90 (Hsp90), M = solubilized membrane protein fraction, H = hydrophilic protein fraction.

Figure 3. Western blots of membrane protein extract prepared from rat liver (Panel A) and rat heart (Panel B). Hydrophobic (membrane protein) fractions and corresponding hydrophilic fractions prepared with Mem-PER Reagents were analyzed by Western blot for VDAC (31 kDa), Flotillin-1 (48 kDa) and Cellugyrin (29 kDa). M: membrane protein fraction; H: hydrophilic fraction.

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Buy Product # Description Pkg. Size Files Price Add 89826 Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit Sufficient lysis and extraction reagents for approximately 50-100 mammalian cell pellet fractions. Kit contains:   Mem-PER Reagent A 10 ml Mem-PER Reagent B 25 ml Mem-PER Reagent C 40 ml Kit $202.00