The Protein Man's Blog | A Discussion of Protein Research

Importance of detergent micelle levels in membrane protein purification

Posted by The Protein Man on Apr 11, 2017 2:00:00 PM
The Protein Man

detergent-solubilization2.jpgDetergents form micelles which can trap hydrophobic molecules into these micelles and allow the extraction of membrane proteins through solubilization.  The “Critical Micelle Concentration” or CMC of a detergent is the concentration of a detergent in which micelles start to form.  Detergents belong to a class of compounds called surfactants.  They are indispensable when working with integral membrane proteins and are able to partition into biological membranes, extract proteins, and maintain protein solubility in solution.  Detergents are useful in a wide variety of applications as well including PAGE, inclusion body solubilization, and lipid raft preparation.  Determining the CMC allows you to choose which detergent may be best for a particular application.  This can be done by a variety of methods including light scattering or measuring the surface tension, both of which can be time consuming and expensive.  A simple method is by plotting optical density of solubilized dye against detergent concentration described by Brigitte Vulliez‐Le Normand and Jean‐Luc Eisele.  G-Biosciences' Optimizer blueBALLS™ uses a similar principle to help determine CMC.

Micellization is a key aspect when considering detergent applications.  Each detergent can be determined by its CMC in which the monomers self-assemble into micelles.  The CMC actually does not occur at a single concentration, but over a narrow range of concentrations.  When the detergent is below the CMC the detergent monomers are free in bulk solution but as more detergent is added above the CMC the detergent monomers will go into micelles.  This is critical so you know how much detergent to add and not in excess which would make the detergent much harder to remove later on downstream.  As a general rule of thumb the detergent concentration of at least 2X CMC and a detergent:protein wt/wt ratio of at least 4:1 is generally used when working with membrane proteins.  When solubilizing proteins from native membranes, it is advisable to work at a detergent concentration of 10:1 detergent:lipid mol/mol ratio.  Therefore, the CMC dictates how much detergent needs to be added to various protein and membrane preparations.

There are several physical and chemical factors that can affect the CMC of any given detergents.  Variations in the hydrophilic head group can affect the CMC such as ionic head groups which can have a higher CMC than nonionic head groups.  Also zwitterionic head groups tend to have smaller CMCs than ionic head groups.  The physical characteristics of the hydrophobic group can also have varying effects on CMC.  For instance, the CMC tends to decrease as the number of carbon atoms in the alkyl chain increases up to approximately 16 to 18 carbons.  Above this point detergents do not form discrete micelles.  Electrolytes tend to reduce the CMC of detergents as well.  For example, the CMC for the anionic detergent SDS is approximately 6mM; however, in the presence of 150mM NaCl, the CMC is reduced to 1.4mM.  The cloud point of a detergent can help to separate water soluble proteins from membrane proteins by an increase in temperature, which favors micelle formation.  The resulting phase separation creates a turbid later in which the micelles precipitated and makes it possible to carry out two-phase water/detergent extractions. 

The CMC is also important in determining which method should be used for detergent removal as detergents may interfere with certain applications.  Detergents with high CMCs are easily removed by dialysis (Tube-O-DIALYZER™).  Detergents with low CMCs are typically removed by adsorption to hydrophobic beads (DetergentOUT™ Tween®) or be removed by column chromatography (DetergentOUT™ GBS10).

For more information on detergents, membrane protein isolation and micelles, check out our other Protein Man Blogs:


Topics: Detergents

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An improved Coomassie Dye based protein assay based on the Bradford Protein Assay. This assay is suitable for the simple and rapid estimation of protein concentration. This assay is based on a single Coomassie dye based reagent. The binding of protein to the dye results in a change of color from brown to blue. The change in color density is proportional to protein concentration. Protein estimation can be performed using as little as 0.5µg protein.


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