The Mammalian Membrane Protein Extraction Kit is a simple, rapid and reproducible method to prepare cellular protein fractions highly enriched in membrane and hydrophobic proteins. This kit reduces sample complexity in order to improve the chances of identifying low abundance proteins, such as membrane proteins, and to simplify proteomic studies. Unlike many procedures used to isolate membrane proteins, this kit does not require the use of ultracentrifugation nor the preparation of density gradients.
The kit is based on the separation of membrane proteins by temperature-dependent phase partitioning using Triton X-114 detergent. The sample is first mixed and homogenized in Permeabilization Buffer. After a brief incubation at 37℃, the sample is centrifuged, producing an upper aqueous phase and a lower detergent-rich phase. Proteins that were anchored to the membrane or proteins containing one or two transmembrane domains are efficiently partitioned to the detergent-rich phase. Cytoplasmic proteins partition into the aqueous phase. An insoluble pellet is also formed and is a source of more complex membrane proteins. Membrane proteins are typically extracted with an efficiency of up to 90%. Cross-contamination of cytosolic proteins into the membrane fractions is usually less than 10%. Extraction of membrane protein can be finished in 120 minutes. And the extracted membrane proteins are compatible with many downstream applications such as SDS-PAGE, BCA protein assay, western blotting, immunoprecipitation and enzymatic activity analysis.
