vbs0065 separation of two model proteins with ion exchange ... · ion exchange chromatography is...
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Separation of two model proteins with Ion Exchange Chromatography (IEC) Michaela Schulze, Ulrike Maschke, Kate Monks; [email protected] Wissenschaftliche Geräte GmbH, Hegauer Weg 38, 14163 Berlin; www.knauer.net
SUMMARY
VBS0065© KNAUER Wissenschaftliche Geräte GmbHAdditional information
Ion exchange chromatography is one of the most widely used FPLC techniques for protein separation and
purification. This application describes an easy separation of two model proteins and explains how ion exchange
chromatography works.INTRODUCTIONIon exchange chromatography (IEC) separates molecules according to type and strength of their charge. The isoelectric point (pI) is the pH where a protein or molecule has no net elec-trical charge. Depending on the pH of the buffer a protein has different surface charges in solution. At a pH above their pI proteins have a negative charge and bind to positively charged resins such as anion exchangers. At a pH below their pI proteins have positive charge and bind to negatively charged cation exchangers. This interaction is used for the separation and purification of various proteins. By using a suitable pH and low salt conditions proteins bind to the resin in the initial step (Fig. 1A). The proteins are mostly separated with a linear salt gradient. The salt ions compete with the proteins for bindings sites. Proteins with weak ionic interactions are the first to elute from the column. In the case of cation exchange chromato-graphy, proteins that are less negatively charged start to elute first (Fig. 1B). With an increase of the salt concentration proteins with stronger ionic interaction elute later from the column (Fig. 1C).
RESULTSα-Chymotrypsinogen A (pI 8.97) and Lysozyme (pI 11.35) are proteins with rather high pI values, which make them ideal candidates for cation exchange chromatography (Fig. 2). Both proteins bind under low salt conditions to the resin. During the wash step that removes potentially unbound protein only a small amount of impurities eluted from the column. α-Chymot-rypsinogen A eluted first from the column due to its lower pI of 8.97 (Fig. 2, peak 1). With an increasing gradient and therefore increasing salt concentration lysozyme eluted as second peak (Fig. 2, peak 2). The eluted proteins were automatically collected by the fraction collector. The salt gradient was monitored by the conductivity monitor (Fig. 2, red signal).
MATERIALS AND METHODIn this application, an AZURA® Bio purification system consisting of AZURA P 6.1L LPG metal-free pump with 10 mL pump head; AZURA ASM 2.1L assistant module with feed pump and two injection valves; an AZURA DAD 2.1L diode array detector with 10 mm, 10 µL flow cell; AZURA CM 2.1S conductivity monitor and Foxy R1 fraction collector was used. Prior to the run the 1 mL cation exchange column (HiTrap Capto S) was equilibrated with 10 mL buffer A (20 mM phosphate buffer pH 7.1). The flowrate for the run was 2 mL/min. 500 µL protein mixture (α-Chymotrypsinogen A 2.5 mg/mL + Lysozyme 2.5 mg/mL) was injected. Subsequently the column was washed with 4 mL buffer A to remove all unbound protein. The two proteins were eluted with a linear gradient over a total volume of 70 mL. The salt concentration increased with the rising concentraion of buffer B (20 mM phosphate buffer pH 7.1 + 1M NaCl) in the gradient from 0 % up to 30 %. Eluting proteins were collected with the fraction collector. The column was regenerated with a high salt wash of 6 mL buffer B following the re-equilibration of the column with 10 mL buf-fer A. The proteins were detected at 280 nm and conductivity signal was recorded to monitor the salt gradient.
CONCLUSIONThis straightforward application illustrates the principle of ion exchange chromatography. Two model proteins with different surface charges eluted under increasing salt concentrations from the cation exchange column. The proteins could be easily separated by cation exchange chromatography with the AZURA® Bio purification system.
Fig. 1 Principle of cation exchange separation A) proteins with different negative charges bind to the cation exchange resin, B) by increasing the salt concentration proteins with a weak negative charge elute first, C) with higher salt concentrations proteins with a strong ne- gative charge elute last
Fig. 2 Chromatogram of the separation of two model proteins with ion exchange chro- matography, blue line - UV 280 nm signal, red line - conductivity signal, 1) peak containing α-Chymotrypsinogen A (2.5 mg/mL), 2) peak containing Lysozyme (2.5 mg/mL)
ADDITIONAL MATERIALS AND METHODS
Separation of two model proteins with Ion Exchange Chromatography (IEC)
Eluent A 20 mM phosphate buffer pH 7.1
Eluent B 20 mM phosphate buffer pH 7.1 + 1 M NaCl
Gradient Volume [mL] % A % B
0 100 0
4 100 0
74 70 30
74.02 0 100
80 0 100
80 100 0
90 100 0
Flow rate 2 mL/min Run time 45 min
Column temperature 25°C Injection mode Full loop
Injection volume 0.5 mL Data rate 2 Hz
Detection wavelength 280 nm Time constant 500 ms
Tab. A1 Method parameters
Instrument Description Article No.
Pump AZURA P 6.1L LPG, metal-free APH64EB
Detector AZURA DAD 2.1L ADC01
Flow cell 10mm, 10µL, Ti, 300bar AMC38
Assistant
AZURA ASM 2.1L Left: Pump with pressure sensor, 50 mL pump head, titanium Middle: 6 port 2 position injection valve, 1/16" connectorsRight: 6 port 2 position injection valve, 1/16" connectors
AYBHECEC
Fraction collector Foxy R1 A59100
Conductivity monitor AZURA CM 2.1S ADG30
Column Capto S, GE Healthcare, HiTrap, 1mL -
Software PurityChrom 3D Option A2654
Tab. A2 System configuration & data
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