Membrane proteins play a key role in the regulation of cellular processes such as proliferation, cytosketetal remodeling, adhesion and apoptosis in eukaryotic cells. Integral membrane proteins are characterized by a hydrophobic domain, which interacts directly with the hydrophobic core of the lipid bilayer. Although integral membrane proteins play a vital role in cellular homeostasis, isolation of these membrane proteins is a formidable task because of their insolubility in most aqueous buffers. The use of strong ionic detergents may disrupt the native structure of integral membrane proteins, thereby rendering them unsuitable for biological assays. Conventional membrane protein fractionation methods are often tedious, inefficacious and suffer from substantial cross contamination of cytosolic proteins into membrane fractions.
The Mem-PER Eurkayotic Membrane Protein Extraction Reagent Kit from Pierce Biotechnologies provides a highly effective two-step method to obtain pure membrane proteins from eukaryotic cells. The first step consists of cell lysis and subsequent solubilization of the membrane proteins using a proprietary reagent (called Reagent A in the kit). The second step involves the extraction of integral membrane proteins by phase-partitioning using a proprietary non-ionic detergent (Reagent B&C). The hydrophobic integral membrane proteins form mixed protein-detergent micelles and accumulate in the detergent layer, from where they can be easily collected. This yields an enriched fraction of pure integral membrane proteins. The biggest advantage of this kit is its simplicity and ease of use. It does not involve labor-intensive procedures like homogenization, sonication or ultracentrifugation steps. Hence, it is very useful for small laboratories and research groups, which do not possess expensive equipment like ultracentrifuges or sonicators. The entire procedure of membrane protein isolation takes about an hour. The membrane proteins obtained with this kit are highly pure and exhibit extremely low cross-contamination from cytosolic proteins. Most importantly, no extra reagents have to be ordered for using the kit; in fact the reagents provided are sufficient for many extractions. The “Instructions” manual provided in the kit contains a detailed description of isolation and extraction protocols accompanied by numerous useful troubleshooting tips.
Two prevalent methods for the analysis of membrane protein fractions are SDS-PAGE and western blotting. Since the membrane protein fractions isolated by this kit are in the detergent layer, it is essential to dilute all of the protein fractions to prevent band and lane distortion during SDS-electrophoresis. One of the major disadvantages of the kit is the dilution of protein samples, which can be a problem if the protein of interest is relatively less abundant. Also, the process of dilution may cause re-precipitation of the hydrophobic integral membrane proteins. We dialyze the membrane protein fraction for 6-8 hours (against Tris-buffered saline containing 0.5% detergent to ensure protein solubility) and find a small fraction of protein precipitates out during dialysis. Pierce recommends the use of another kit namely the PAGEprepTM Protein Clean-up and Enrichment kit to remove detergent from the samples. I wish they had provided the reagent within this kit, which would at least partially resolve this problem. Nevertheless, I would strongly recommend the Mem-PER Eukayotic Membrane Protein Extraction Reagent Kit from Pierce Biotechnologies as an extremely valuable and efficient tool for obtaining pure integral membrane proteins from cells, suitable for most cellular assays and biochemical experimental systems.
Piyali Dasgupta, Ph.D.
Post Doctoral Fellow
Moffitt Research Center