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Purification of Proteins Using Displacement Chromatography 83
proteins (feed) and displacer, respectively. Otherwise, a system that will include
an HPLC pump, an injection valve (a valve that can accommodate two
different injection loops such as a Model C10W 10 port valve (Valco) will
be preferable), UV-Vis detector, fraction collector and data recorder can be
assembled.
2.5.3. Methods
The methods described here are common for both cases.
1. Equilibrate the column with 5–10 column volume (CV) of the equilibration buffer.
If preferred, completion of equilibration can be checked via an in-line conductivity
meter or pH meter (AKTAExplorer 100) or by simply collecting the effluent and
checking the pH and conductivity with stand alone detectors.
2. Prepare the injection valve to first load the protein mixture and then the displacer
solution on to the column. Make sure the lines from the protein mixture and
displacer solution are primed with the corresponding solutions.
3. Start loading the protein mixture onto the column by switching the valve position
to the loop (line) that contains the protein mixture. Start monitoring the column
effluent at 280 nm. If an increase in absorbance is detected, start the fraction
collector to collect the effluent (200–400 μL fractions can be collected).
4. As soon as loading of the protein mixture is over, switch the injector valve
position to the loop (line) that contains the displacer solution. Monitor the effluent
absorbance. When the absorbance at 280 nm starts increasing (indicating the
elution of proteins), start to collect fractions.
5. Once it is established that displacer breakthrough has occurred (see Note 6),
regenerate the column with 10–20 CV of regeneration buffer. Collect fractions
during regeneration for analysis (large fractions such as 5–10 mL will be sufficient)
for further analysis. Re-equilibrate the column as described in step 1.
6. Analyze the fractions collected during the displacement experiments. The protein
mixture -lactoglobulin A and B can be analyzed using anion exchange chromatography
(Source 15Q) at isocratic conditions at a flow rate of 1 mL/min. The mobile
phase used is 50 mM Tris–HCl + 130 mM NaCl buffer at a pH of 7.5. The fractions
are diluted threefold to fivefold and 5-μL samples were injected. Column effluent
is monitored at 235 nm. Saccharin can be assayed using size exclusion chromatography.
The fractions are diluted threefold, and 5-μL samples are injected. The
column effluent is monitored at 254 nm. For analysis of SOS, a phenol-sulfuric
acid assay can be used (see Note 6) (35).
7. Construct the displacement chromatogram based on the fraction analysis and
determine the purity and yield of the protein components of interest. If the
resolution and purity of the protein components are sufficient, pool the fractions
based on the fraction analysis. If separation and/or yield are not satisfactory,