Preparative Separation of Proteins Using Centrifugal Precipitation Chromatography Based on Solubility in Ammonium Sulfate Solution

Abstract

A preparative modification of the centrifugal precipitation chromatography (CPC) is described. The sample‐loading capacity is improved in the present system by the use of convoluted tubing containing dialysis tubing instead of a dialysis membrane placed between a pair of disks equipped with mirror‐imaged spiral grooves as in the original design. The system uses, basically, the same principle of as the original CPC, in that a concentration gradient of precipitant is generated under a centrifugal force field. The protein sample injected into the CPC column is exposed to an increasing concentration of the precipitant where it precipitates at various portions of the column according to its solubility. The gradient is then gradually lowered so that the sample undergoes dissolution and precipitation many times within the column; the proteins finally elute from the column according to their solubilities. A basic study was performed using this machine to separate human albumin and 3‐globulin using ammonium sulfate (AS) as precipitant. Preliminary results indicate that this method can separate 500 mg of protein.

Keywords:

• Centrifugal precipitation chromatography
• Protein separation and purification
• Ammonium sulfate gradient precipitation
• Preparative separation
• Dialysis tubing
• Convoluted tubing

Acknowledgments

The authors are indebted to Dr. Fuquan Yang of the Laboratory of Biophysical Chemistry, NHLBI, National Institutes of Health, for his tutoring of various techniques. They also thank to Dr. Henry M. Fales of Laboratory of Biophysical Chemistry, NHLBI, National Institutes of Health, for editing the manuscript.