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JoVE Encyclopedia of Experiments
Encyclopedia of Experiments: Biological Techniques

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Cation Exchange Chromatography: A Technique to Isolate Target Recombinant Protein from Insect Cell Lysate Based on Net Surface Charge

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Cation exchange chromatography facilitates protein separation from mixtures based on their net surface charge.

For target signal transducer protein isolation from insect cell protein lysate, obtain a dialyzed target protein lysate containing other contaminating proteins. Add sodium chloride-containing buffer to reduce lysate viscosity and improve protein solubility, which helps prevent chromatographic column clogging during run.

Additionally, buffer pH below the target protein's isoelectric point, pI, at which the protein has zero net charge, facilitates binding of excess hydrogen ions to negative carboxylate groups, making the target protein positively charged.

Assemble the chromatographic column containing crosslinked agarose beads coupled to negatively-charged groups. Add binding buffer to equilibrate the column and maximize column-protein interactions. Load the diluted lysate onto the equilibrated column.

More positively-charged target proteins interact and bind tightly to the negatively-charged groups of the column than less positively-charged contaminating proteins.

Wash the column with buffer to remove unbound negatively charged contaminating proteins. Subsequently, pass elution buffer, while gradually increasing its ionic strength.

At low ionic strength, there is minimal competition between the positive ions and proteins for the negative groups in the column, eluting weakly bound, less positively-charged contaminating proteins. However, as the ionic strength is increased, salt ions compete with tightly bound target signal transducer proteins and reduce interactions between the proteins and the column, eluting them as a purified fraction.

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