Centrifugal precipitation chromatography.
Centrifugal precipitation chromatography separates analytes according their solubility in ammonium sulfate (AS) solution and other precipitants. The separation column is made from a pair of long spiral channels partitioned with a semipermeable membrane. In a typical separation, concentrated ammonium sulfate is eluted through one channel while water is eluted through the other channel in the opposite direction. This countercurrent process forms an exponential AS concentration gradient through the water channel. Consequently, protein samples injected into the water channel is subjected to a steadily increasing AS concentration and at the critical AS concentration they are precipitated and deposited in the channel bed by the centrifugal force. Then the chromatographic separation is started by gradually reducing the AS concentration in the AS channel which lowers the AS gradient concentration in the water channel. This results in dissolution of deposited proteins which are again precipitated at an advanced critical point as they move through the channel. Consequently, proteins repeat precipitation and dissolution through a long channel and finally eluted out from the column in the order of their solubility in the AS solution. The present method has been successfully applied to a number of analytes including human serum proteins, recombinant ketosteroid isomerase, carotenoid cleavage enzymes, plasmid DNA, polysaccharide, polymerized pigments, PEG-protein conjugates, etc. The method is capable to single out the target species of proteins by affinity ligand or immunoaffinity separation.
Ito
Yoichiro
Y
Bioseparation Technology Laboratory, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1762, USA. itoy@nhlbi.nih.gov
Qi
Lin
L
D000645:Ammonium Sulfate / Q000737:chemistry; D001798:Blood Proteins / Q000302:isolation & purification; D002498:Centrifugation / Q000295:instrumentation* / Q000379:methods*; D011232:Chemical Precipitation; D002846:Chromatography, Affinity / Q000379:methods*; D004867:Equipment Design; D006801:Humans; D007163:Immunosorbent Techniques; D011506:Proteins / Q000302:isolation & purification*
D001798:Blood Proteins; D011506:Proteins; D000645:Ammonium Sulfate