scholarly journals Membrane protein extraction and purification using partially-esterified SMA polymers

Author(s):  
Olivia P. Hawkins ◽  
Christine Parisa T. Jahromi ◽  
Aiman A. Gulamhussein ◽  
Stephanie Nestorow ◽  
Taranpreet Bahra ◽  
...  
Soft Matter ◽  
2021 ◽  
Author(s):  
Marcelo Caparotta ◽  
Marcelo Puiatti ◽  
Diego Masone

Styrene-maleic acid copolymers have become an advantageous detergent-free alternative for membrane protein isolation. Since their discovery, experimental membrane protein extraction and purification by keeping intact their lipid environment has become...


2003 ◽  
Vol 2003 (4) ◽  
pp. 249-255 ◽  
Author(s):  
M. Walid Qoronfleh ◽  
Betsy Benton ◽  
Ray Ignacio ◽  
Barbara Kaboord

The human proteome project will demand faster, easier, and more reliable methods to isolate and purify protein targets. Membrane proteins are the most valuable group of proteins since they are the target for 70–80% of all drugs. Perbio Science has developed a protocol for the quick, easy, and reproducible isolation of integral membrane proteins from eukaryotic cells. This procedure utilizes a proprietary formulation to facilitate cell membrane disruption in a mild, nondenaturing environment and efficiently solubilizes membrane proteins. The technique utilizes a two-phase partitioning system that enables the class separation of hydrophobic and hydrophilic proteins. A variety of protein markers were used to investigate the partitioning efficiency of the membrane protein extraction reagents (Mem-PER) (Mem-PER is a registered trademark of Pierce Biotechnology, Inc) system. These included membrane proteins with one or more transmembrane spanning domains as well as peripheral and cytosolic proteins. Based on densitometry analyses of our Western blots, we obtained excellent solubilization of membrane proteins with less than 10% contamination of the hydrophobic fraction with hydrophilic proteins. Compared to other methodologies for membrane protein solubilization that use time-consuming protocols or expensive and cumbersome instrumentation, the Mem-PER reagents system for eukaryotic membrane protein extraction offers an easy, efficient, and reproducible method to isolate membrane proteins from mammalian and yeast cells.


eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Claudia C Schmidt ◽  
Vedran Vasic ◽  
Alexander Stein

In endoplasmic reticulum-associated protein degradation (ERAD), membrane proteins are ubiquitinated, extracted from the membrane, and degraded by the proteasome. The cytosolic ATPase Cdc48 drives extraction by pulling on polyubiquitinated substrates. How hydrophobic transmembrane (TM) segments are moved from the phospholipid bilayer into cytosol, often together with hydrophilic and folded ER luminal protein parts, is not known. Using a reconstituted system with purified proteins from Saccharomyces cerevisiae, we show that the ubiquitin ligase Doa10 (Teb-4/MARCH6 in animals) is a retrotranslocase that facilitates membrane protein extraction. A substrate’s TM segment interacts with the membrane-embedded domain of Doa10 and then passively moves into the aqueous phase. Luminal substrate segments cross the membrane in an unfolded state. Their unfolding occurs on the luminal side of the membrane by cytoplasmic Cdc48 action. Our results reveal how a membrane-bound retrotranslocase cooperates with the Cdc48 ATPase in membrane protein extraction.


2009 ◽  
Vol 2009 ◽  
pp. 1-10 ◽  
Author(s):  
Siun Chee Tan ◽  
Beow Chin Yiap

Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples; the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination.


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