Once a membrane protein is extracted from membrane fragments, the target protein can be further purified to high homogeneity. Common chromatographic techniques, such as affinity chromatographic, size exclusion chromatographic (SEC), and ionic exchange chromatographic (IEC), can be applied to protein purification. However, the following facts need to be considered:

1.Sufficient amount of a detergent is required for solubilizing integral membrane proteins and preventing aggregation of the proteins.

2.Due to the amphipathic nature of membrane proteins, protein separation methods based on hydrophobicity, such as phenyl-sepharose chromatography and reverse-phase chromatography, are normally inappropriate for membrane protein purification.

3.Ionic detergents, such as bile salts or deoxycholates, are not suitable for ion exchange chromatography (IEC) because they contain charged groups that compete with the analyte for binding to the ionic resin. Non-ionic or zwitterionic detergents can be used for charge-based preparation methods, including ion exchange chromatography and preparative electrophoresis.

4.Detergents with sugar moieties may interfere with lectin-based chromatography. For example, hexaglycoside detergents interfere with the affinity chromatography that consists of ConA [binding to dextran mannose] and wheat germ lectins.

5.Detergent micelles always have an unneglectable size, a detergent-solubilized membrane protein shows a larger apparent molecular weight than its actual molecular weight during purification by size-exclusion chromatography (SEC). For example, Trtion X-100 and DDM can increase the molecular weight of dissolved membrane proteins by 60 to 100 kDa. Therefore, a detergent-solubilized protein is usually collected at a high molecular weight fraction in SEC.

6.The detergents, especially those ionic and with large micelle sizes, can mask the charges of membrane proteins. Therefore, the ionic exchange chromatography for membrane protein preparation may not be as effective as it is for soluble proteins.

7.In general, affinity chromatography is the most effective method for purifying multi-pass membrane proteins. In contrast to other chromatography methods such as IEC that is sensitive to the ionic strength and SEC that has restrictions on sample volume, affinity chromatography does have such limitations and can be used at any purification step, including sample concentration and salt replacement.