pH-gradient ion-exchange chromatography: an analytical tool for design and optimization of protein separations.
This work demonstrates that a highly linear, controllable and wide-ranged pH-gradient can be generated through an ion-exchange chromatography (IEC) column. Such a pH-gradient anion-exchange chromatography was evaluated with 17 model proteins and found that acidic (pI<6) and basic (pI>8) proteins elute roughly at their pI, whereas neutral proteins (pI 6-8) elute at pH 8-9 regardless their pI values. Because of the flat nature of protein titration curves from pH approximately 6 to approximately 9, neutral proteins indeed exhibit nearly zero net charge at pH approximately 9. The elution-pH in pH-gradient IEC or the titration curve, but not the pI, was identified as the key parameter for pH optimization of preparative IEC in a fast and rational way. The pH-gradient IEC was also applied and found to be an excellent analytical tool for the fractionation of crude protein mixtures.
Department of Biotechnology, Delft University of Technology, Delft, The Netherlands. t.ahamed@tudelft.nl
Tangir
T
Ahamed
Beckley K
BK
Nfor
Peter D E M
PD
Verhaert
Gijs W K
GW
van Dedem
Luuk A M
LA
van der Wielen
Michel H M
MH
Eppink
Emile J A X
EJ
van de Sandt
Marcel
M
Ottens
D000837:Anion Exchange Resins / Q000737:chemistry; D002852:Chromatography, Ion Exchange / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D018892:Proton-Motive Force; D015203:Reproducibility of Results
D000837:Anion Exchange Resins; D011506:Proteins