The Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit enables small-scale solubilization and enrichment of integral membrane proteins and membrane-associated proteins in a simple reagent-based procedure.
The Mem-PER Plus Kit effectively isolates membrane proteins from cultured cells and tissues using a simple benchtop microcentrifuge procedure. Integral membrane proteins having one or two membrane-spanning domains are effectively solubilized and isolated from cytosolic proteins. The kit makes possible the extraction and selective enrichment of integral and attached membrane proteins from cultured mammalian cells, as well as from hard and soft mammalian tissues. The Mem-PER Plus Kit has many advantages compared to our original Mem-PER Kit. The protocol is simpler, and the fractions are directly compatible with many downstream applications such as SDS-PAGE, Western Blotting, BCA, immunoprecipitation, and amine-reactive protein labeling techniques.
- Extraction and isolation – produces minimal cross-contamination (typically less than 10%) of cytosolic protein into the membrane protein fraction
- Cells or tissues – effective for extraction from cultured mammalian cells and mammalian tissues
- Downstream compatibility – analyze membrane protein extracts by SDS-PAGE, Western blotting, immunoprecipitation and protein assays
- Fast and simple – isolation of membrane proteins in approximately one hour
- No special equipment required – only a benchtop microcentrifuge, tubes, homogenizers, and pipettors are needed
Kit contains a cell wash solution, permeabilization buffer, and solubilization buffer.
Traditional methods for isolation of membrane protein are tedious and time-consuming, and they require gradient separation and expensive ultracentrifugation equipment. The Mem-PER Plus Membrane Protein Extraction Kit is for the enrichment of integral membrane proteins and membrane associated proteins from cultured mammalian cells or tissue using a mild detergent-based selective extraction protocol. The use of selective detergent extraction eliminates the hassle of phase separation based on hydrophobicity, allowing better reproducibility and higher throughput.
|Protocol summary for the Mem-PER Plus Membrane Protein Extraction Kit.
The cells are first permeabilized with a mild detergent, allowing the release of soluble cytosolic proteins, after which a second detergent solubilizes membrane proteins. Extraction efficiencies and yields will vary depending on cell type as well as the number of times the integral membrane protein spans the lipid bilayer. Membrane proteins with one or two transmembrane domains are typically extracted with an efficiency of up to 90%. Cross-contamination of cytosolic proteins into the membrane fraction is usually less than 10%.
|Membrane protein yields from common cell lines obtained with two popular extraction kits. Membrane proteins were isolated using the Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit and the EMD Millipore ProteoExtract™ Native Membrane Protein Extraction Kit (Part No. 444810), following the respective protocols. Membrane protein yield was determined by BCA Protein Assay (Part No. 23225) with BSA standards (Part No. 23209).
|Isolation and enrichment of membrane proteins from different tissues. Membrane proteins were isolated from frozen mouse heart or brain (30mg) following the Mem-PER Plus Membrane Protein Extraction Kit protocol. Membrane and cytosolic fractions (10μg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Western blots were done using the Thermo Scientific Fast Western Rabbit Dura Kit (Part No. 35071) and primary antibodies diluted 1:1000. Images were generated using the Thermo Scientific myECL Imager (Part No. 62236).
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