SEC/reversed-phase separation of E. coli proteins.
Although many chromatographic modes can be coupled for the multidimensional separation of a complex mixture, a very favorable combination is that of size-exclusion chromatography (SEC) and reversed-phase chromatography. The separation mechanisms are largely orthogonal and the mobile phases are compatible. The use of a retentive second dimension allows trapping of specific fractions from the SEC separation and transferring them to the reversed-phase separation. One of the advantages of multidimensional chromatography is that it is scalable in terms of automation. In a completely manual system, fractions eluting from a first dimension can be manually collected and injected into a second separation dimension. In an automated system, fraction transfer can be accomplished through automated valving. The following protocol illustrates this approach, with both a manual method and an external column switching method integrated into an automated high-performance liquid chromatography (HPLC) method. As an illustration, soluble proteins from Escherichia coli are separated.
Apffell
Alex
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D002850:Chromatography, Gel / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000302:isolation & purification*
D029968:Escherichia coli Proteins