In the final step of purification of Taq DNA Technique: Cation exchange chromato

Question

In the final step of purification of Taq DNA
Technique: Cation exchange chromatography
Resin: CM-Sephadex (negative charged)
Taq sample ( Taq DNA, PEI, some impurities)
What is the stationary phase used during chromatography?
What was the mobile phase?
Did Taq DNA polymerase locate in the stationary or the mobile phase in each process: binding step, low salt washing step, elution step ???
In washing step, why we add the buffer containing 0.05 M KCl to the resin?
In elution, why we add the buffer containing 0.2 M KCl to the resin?

Explanation / Answer

a. The stationary phase is the CM-Sephadex, which is negatively charged and pre-equilibrated with the buffer that is used in mobile phase. Thus it captures positively charged analytes from the mobile phase.

b. The mobile phase is the Taq sample, mixture of Taq DNA, PEI (Polyethyleneimine) and some impurities, which is dissolved in the low ionic strength buffer.

c. During initial binding, Taq DNA polymerase is observed in the stationary phase, because DNA Pol binds to the negatively charged resins.

At low salt washing step, DNA pol still remain attached to the stationary phase, as less number of salt ion is available which can compete with the proteins for the functional groups of the stationary phase. In this step, some impurities which are loosely associated with the negatively charged resin, are eliminated.

At the elution step, DNA Pol is definitely in the mobile phase, so that it can be eluted from the column.

d. The aim of the washing step is to eliminate the impurities, which possess less positive charge than the protein molecule. The low salt concentration (0.05 M KCl) is used, as the present salt ions are capable to compete with these impurities for a functional groups of the stationary phases, but not sufficient to compete against the protein molecules. In the elution step, high concentration of salt (0.2 M KCl) ensures complete removal of protein molecules from the stationary phase and protein begins to elute from the column in order to increasing charge

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