Membrane Protein Isolation and Purification With Phase Separation

It is interesting to note that while detergent-solubilized membrane proteins can be purified and concentrated through phase separation (which is a simple, efficient and cost-effective method that can be used for this purpose), most membrane protein scientists do not use this method or even know that such methods exist. To better understand and appreciate how this method works, here are some things you definitely need to know.

Phase Separation: Understanding the Basics

In a nutshell, phase separation (also known as cloud point extraction) refers to the state wherein the micelles in an aqueous solution become immiscible with water and form large aggregates that will separate from the water phase. This condition is usually achieved upon the addition of detergents or when the temperature or salt concentration of the solution is altered.

Under such conditions, most of the detergent will partition into the detergent-rich phase but will still contain a small amount of water. The detergent-rich phase can either be clear or turbid, depending on the detergent used and the existing buffer condition, and may be found either on top or below the detergent-poor phase.

Considering these things, phase separation can be an excellent addition, and may even be considered as a powerful alternative, to chromatography-based purification protocols since it can be used directly on solubilized membranes to separate the membrane proteins from the soluble proteins and other hydrophilic impurities. In addition, it can also be used as a polishing step during the later stages of purification.

The Role of Detergents in Phase Separation

Detergents are used in phase separation due to their unique molecular structure. Detergents are amphipathic molecules, meaning they have a polar or charged headgroup and an extended hydrophobic hydrocarbon chain. These molecules are soluble in water at very low concentrations, but once the detergent concentration goes above the so-called critical micelle concentration (CMC), the molecules will form aggregates with a very narrow size distribution (micelles).

The type of detergent, ionic strength and the temperature of the detergent solution dictate the size of the micelles formed. The aggregation number or the number of detergent molecules per micelle can range from 2 to 3 for sodium cholate to about 140 for Triton® X-100.

As a rule, the CMC of ionic detergents decreases with increasing salt concentrations (although it is hardly affected by temperature). On the other hand, the presence of salts has very little effect on the CMC of non-ionic detergents. However, it is observed to increase with increasing temperature. The presence of impurities, pH and pressure can also influence the CMC and the size of the micelle. 

Some of the detergents that have been successfully used for membrane protein purification include the following:

• Triton ® (Triton X-100 and Triton X-114)
• Tween ® (Tween 20 and Tween 80)
• Polyoxyethylene Glycol (PEG) monoether detergents
• Glycosidic detergents such as alkyl β-glucosides and alkyl β-maltosides
• Anionic detergents such as sodium dodecyl sulfate (SDS)
• Other Nonionic detergents such as N,N-dimethyldodecylamine-N-oxide (DDAO) and Digitonin
• Cationic detergents such as dodecyltrimethylammonium bromide (DTAB or C₁₂TAB)
• Zwitterionic detergents

The Pros and Cons of Using Phase Separation in the Purification Process

There are a number of benefits that you can get by using phase separation in the purification of membrane proteins. Here are some of them:

• The protocols are simple to use.
• It does not require complex laboratory equipment.
• It can easily be scaled up to large volumes and applied to industrial processes.
• The method is compatible with most chromatographic methods.
• It efficiently removes hydrophilic compounds from the solution.

However, there is also a downside to using this method. Since it requires high detergent concentrations, it may negatively affect the stability of your protein of interest. The presence of high detergent concentrations may also interfere with biochemical assays and binding processes so you may need to perform dialysis, detergent absorption or gel filtration to remove any excess detergent from the solution.