Cation Exchange Chromatography: A Technique to Isolate Target Recombinant Protein From Insect Cell Lysate Based on Net Surface Charge
Trascrizione
Prepare a 20-milliliter cation exchange column by washing it with three column volumes of Buffer G, then with three column volumes of Buffer F. Next, dilute 20 milliliters of the dialyzed sample to a final sodium chloride concentration of 100 millimolar, by adding 20 milliliters of Buffer E, and apply the sample to the exchange column.
It is critical to dilute the small amount of the sample instead of all at once, and the diluted samples should be applied to the column immediately after dilution to limit precipitation of the protein.
Continue to load the column with freshly diluted sample as the previous dilution nears the end of the load. Then, wash the column to baseline absorbance 280 with Buffer F, which typically required three column volumes.
Elute the protein from the column with a 400-milliliter gradient from Buffer F to 65% Buffer G, collecting the eluate in 6-milliliter fractions. Once the gradient is complete, continue washing the column for an additional 1.5 column volumes of 65% Buffer G.
