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

Detergents 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.

Affinity chromatography is the most popular and widely used method for purification of biomolecules including proteins, antibodies, lectins, carbohydrates, nucleic acids and glycoproteins. Affinity purification is based upon specific surface interactions among biological molecules such as antigen-antibody, enzyme-ligand etc. These specific surface interactions enable the purification of molecules from 1000 to 10,000 fold in a single step. In affinity chromatography, the ligand, antigen or one of the interacting molecules is covalently bound to the matrix and is used as a bait to fish out the target protein from any complex pool of proteins including cell or tissue lysate.

Extracting proteins from plant and animal sources are two different things. While animal proteins can easily be extracted, working with plant proteins can be quite a challenge. Why? Plant proteins are more difficult to extract since they are protected by a rigid cell wall that contains several interfering agents (i.e proteases).

As mentioned in our earlier posts, extracting, separating and identifying proteins from plant samples present a unique challenge since they contain significant amounts of proteases and other compounds that may interfere with the process. Generally, there are three critical aspects for successful protein separation: