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Determining the structure of a membrane protein often requires the use of many orthogonal crystallization methods. Of the membrane protein structures determined by X-ray crystallography in 2015, the majority were determined by vapor diffusion, followed by lipidic cubic phase, and lastly bicelle crystallization (61%, 35%, 4%, respectively - see February 2016 newsletter). Vapor diffusion experiments are typically carried out in detergent micelles, which form a taurus around the transmembrane region of the protein and allow for type II crystal packing. Lipidic cubic phase crystallization, first described in 1996⁽¹⁾, utilize a continuous lipid (typically monoolein) bilayer, and allow for the formation of type I crystals. Membrane proteins reconstituted into bicelles typically crystallize with type I packing.
 
Bicelles as a tool for membrane protein biochemistry were first described in 1990⁽²⁾, and studies showing the functionality of a reconstituted membrane protein in bicelles came from the lab of Chuck Sanders (Vanderbilt University) in 2000⁽³⁾. Two years later marked the first time bicelles were utilized in membrane protein crystallization with the structure of bacteriorhodopsin⁽⁴⁾.
 
Bicelles are formed by the mixing of a phospholipid and a detergent at specific ratios, and have very unique temperature dependent phase properties which are useful for crystallization experiments ^{(2, 5, 6, 7)}. The phospholipid typically used to form bicelles is DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine – D514), while the most commonly used detergent is CHAPSO (3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate – C317); however DHPC (1,2-Dihexanoyl-sn-Glycero-3-Phosphocholine - D606) has also been used. The ratio of phospholipid to detergent in bicelle mixtures can vary, but the most commonly used ratio is 2.8:1. At lower temperatures, bicelles mixtures are liquid, and can easily be manipulated with pipettes or liquid handling robots, while at higher temperatures, the bicelles form a clear gel.
 
In total, over twenty membrane protein structures, from most major functional classes have been determined from bicelles (see the table below for a selection of structures determined from bicelles – data adapted from⁽⁵⁾).  

In order to provide options for bicelle crystallization, Anatrace is excited to offer pre-mixed bicelle solutions of the two most commonly used lipid – detergent combinations. Anatrace pre-mixed bicelles are available as 250 µl of 30% stock solutions of either 2.8:1 DMPC:CHAPSO (D606:C317 BIC MIX 0.25 ML) or 2.8:1 DMPC:DHPC (D606:D514 BIC MIX 0.25 ML).  For initial crystallization screening, we recommend screening between 5% - 8% bicelles. To reconstitute a protein into bicelles, protein is mixed with the bicelle solution in a 4:1 ratio⁽⁷⁾. For example, to create 100 µl of a 5% protein-bicelle mixture, the 30% stock bicelle solution is first diluted with H₂O to 25% (16.7 µl 30% bicelles + 3.3 µl H₂O = 20 µl 25% bicelles), and then added to 80 µl of concentrated protein solution. The reconstituted protein in bicelles can then be used to setup crystallization experiments in either hanging drop, sitting drop, or the Microlytic Crystal Former plates. A full protocol for protein reconstitution and crystallization will be available on the bicelles product page on the Anatrace website.