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Mem-PER Eukaryotic Membrane Protein Extraction Kit


Extracts membrane-associated proteins without gradient ultracentrifugation.

Mem-PER Membrane Protein Extraction ReagentThe Thermo Scientific Mem-PER Eukaryotic Membrane Protein Extraction Kit enables small-scale solubilization and enrichment of hydrophobic membrane-associated proteins in a simple reagent-based procedure.

Note:
This product will be discontinued in Spring 2014.
A new, improved version of this kit is now available:
Mem-PER Plus Membrane Protein Extraction Kit
 

Traditional methods for isolation of membrane protein are tedious and time-consuming, and they require gradient separation and expensive ultracentrifugation equipment. The Mem-PER Kit effectively isolates membrane proteins from cultured cells and tissues using a simple benchtop microcentrifuge procedure. Hydrophobic proteins having three or fewer membrane-spanning domains are effectively solubilized and isolated from hydrophilic (cytoplasmic) proteins using the three-reagent system. The kit makes possible the 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.

Highlights:

  • Extraction and isolation – produces minimal cross-contamination (typically less than 10%) of hydrophilic protein into the hydrophobic (membrane) protein fraction
  • Cells or tissues – effect for extraction from cultured mammalian and yeast cells and mammalian tissues
  • Downstream compatibility – analyze membrane protein extracts by SDS-PAGE, Western blotting, and protein assays
  • Fast and simple – isolation membrane proteins in approximately one hour
  • No special equipment required – only a benchtop microcentrifuge, tubes and pipettors are needed

Product Details:

Mem-PER Membrane Protein Extraction Reagent Protocol for Yeast and Mammalian Cells
Protocol summary for the Mem-PER Membrane Protein Extraction Kit. Preparation of mammalian membrane protein extracts using Mem-PER Mammalian Membrane Protein Extraction Kit can be done quickly and efficiently. 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.

Validation experiments demonstrate that the Mem-PER Kit is highly efficient in the extraction of integral membrane proteins containing one or two transmembrane spanning domains. Results were consistent among three different cell lines tested: NIH 3T3, HeLa and C6. The integral membrane protein flotillin, containing two transmembrane domains, was extracted with an efficiency of approximately 50% from the three cell lines. Extraction of cytochrome oxidase subunit 4 (Cox4), containing one transmembrane domain, was 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 (less than 10%).

Acetylcholinesterase (AchE), a peripheral protein, and Heat shock protein 90 (Hsp90), a cytosolic protein, partitioned into the hydrophilic fraction with an efficiency greater than 90%. The remaining 10% or less that remained 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 less than 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. Protein assays, subsequent purification, etc. can be performed following removal of Reagent C through dialysis. To effectively remove Reagent C and simultaneously maintain protein solubility, perform dialysis overnight at 4°C with a buffer that includes 0.5% CHAPS detergent. Quantification of extracted membrane proteins is possible with the Thermo Scientific Pierce Micro BCA Protein Assay Kit (Part No. 23235) and typically results in approximately 250µg of total protein from 5 million C6 cells. The total amount of protein obtained will vary depending on the cell line.


Yeast membrane protein solubilization and isolation.
 Yeast membrane protein solubilization and isolation. The Thermo Scientific Mem-PER Kit was used to extract membrane proteins from Saccharomyces cerevisiae strain EGY-194. Wet cell paste (10 to 15mg) was vortexed for 10 minutes at room temperature in 80μL of Mem-PER Reagent A and 100 to 150mg of acid-washed glass beads (405-600μn dia.) to disrupt the yeast cell wall. After pelleting the beads by centrifugation, the cell lysate was transferred to a new tube and kept on ice. The beads were washed with 720μL of Mem-PER Reagents B and C, and the wash was then combined with the cell lysate. Finally, the solution was incubated on ice for 30 minutes. Samples were resolved on 4-12% Bis-Tris gels and examined by Western blot using target-specific antibodies and chemiluminescent substrate. 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).


Effective extraction and partitioning of membrane and cytoplasmic proteins.
Effective extraction and partitioning of membrane and cytoplasmic proteins. Proteins from three cell lines were solubilized and extracted using the Thermo Scientific Mem-PER Kit. Each set of membrane (M) and hydrophilic (H) protein fractions were normalized to one another and analyzed by Western blot for four proteins from the cellular locations noted. The Thermo Scientific Pierce SDS-PAGE Sample Prep Kit (Part No. 89888) was used to remove the detergent from the membrane fraction before electrophoresis analysis of COX4. A negligible amount of protein was found in all debris fractions. AchE is acetylcholinesterase; COX4 is cytochrome oxidase subunit 4; hsp90 is heat shock protein 90.


Partitioning of Proteins with the Mem-PER Kit
Cell
Type
Fraction Flotillin
(2-spanner)
Cox 4
(1-spanner)
AchE
(peripheral)
Hsp90
(cytosolic)
NIH 3T3 Membrane
Hydrophilic
45
55
90.8
9.2
1.7
98.3
6.8
93.2
HeLa Membrane
Hydrophilic
48.4
51.6
89.1
10.8
4.1
95.9
15.5
84.4
C6 Membrane
Hydrophilic
56
44
94.5
5.5
6.4
93.6
10.6
89.4



Membrane protein extraction from rat liver and heart tissue.
 

Membrane protein extraction from rat liver and heart tissue. Hydrophobic (membrane protein, M) fractions and corresponding hydrophilic (H) fractions were prepared from rat liver (A) and rat heart (B) tissue using the Thermo Scientific Mem-PER Kit. Extracts were analyzed by Western blot for VDAC (31 kDa), cellugyrin (29 kDa) and flotillin-1 (48 kDa) using specific antibodies and chemiluminescent substrate.

References:

  1. Fukuchi, J., et al. (2004). Androgenic Suppression of ATP-binding Cassette Transporter A1 Expression in LNCaP Human Prostate Cancer Cells. Cancer Res. 64: 7682–7685,
  2. Stefano, G.B., et al. (2003). Estrogen Signaling at the Cell Surface Coupled to Nitric Oxide Release in Mytilus edulis Nervous System. Endocrinology 144(4): 1234–1240
  3. Wojnar, P., et al. (2003). Antisense Down-regulation of Lipocalin-interacting Membrane Receptor Expression Inhibits Cellular Internalization of Lipocalin-1 in Human NT2 Cells. J. Biol. Chem. 278(18): 16209–16215
  4. Ohlendieck, K. (1996). Protein Purification Protocols; Methods in Molecular Biology, Humana Press Inc., Totowa, NJ. Vol 59: 293-304.
  5. Morre, J. and Morre, D. (1989). Preparation of Mammalian Plasma Membranes by Aqueous Two-Phase Partitioning. Biotechniques 7(9): 946-958.
  6. Lenstra, J. A. and Bloemendal, H. (1983). Topography of the total protein population from cultured cells upon fractionation by chemical extractions. Eur. J. Biochem. 135: 413-423.

Related Resources:

Review of cell lysis and protein extraction
Review of cell fractionation and organelle isolation

Related Products:

Mem-PER Plus Membrane Protein Extraction Kit


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
89826 Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit
Formulation: Multi-component kit
Sufficient For: 60 to 80 samples each containing 5 million cells, 15mg yeast cell paste or 20mg tissue

Kit Contents:
Mem-PER Reagent A, 10mL
Mem-PER Reagent B, 25mL
Mem-PER Reagent C, 40mL
75mL kit Product Instructions for product #89826 Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit MSDS for product #89826 Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit Certificate of Analysis for product #89826 Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit $253.00

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Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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