產品名稱：Mem-PER Membrane Protein Extraction Reagent
貨號：Pierce 89826
規格：kit
The 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. 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.
Product Details:
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 approximay 50% from the three cell lines. Extraction of cytochrome oxidase subunit 4 （Cox4）, containing one transmembrane domain, was even more efficient with approximay 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 approximay 250µg of total protein from 5 million C6 cells. The total amount of protein obtained will vary depending on the cell line