Calcium-Dependent Hydrophobic Interaction Chromatography: A Technique to Purify Calcium-Binding Proteins Based on Hydrophobic Interactions
Transcript
To continue the protein purification, after the protein dialysis, add calcium chloride to the sample to a final concentration of 25 millimolar, which will facilitate binding of S100A12 to the resin in the next step. Then, filter the sample through a filter with a pour size of 0.45 microns.
Equilibrate HIC buffers and samples to 4 degrees Celsius. Next, connect column buffers HIC-A and B and the column, to the system and adjust the parameters.
Start the method to equilibrate the column with 1 to 2 column volumes of buffer HIC-A, and load the sample, and the 'wash unbound sample block' when the UV signal reaches baseline level again.
Then, start elution with a calcium chelator-containing buffer EDTA. Collect 2 milliliters of peak fractions during elution.
