Zone precipitation chromatography: its use in the isolation of different collagen types

Zone Precipitation Chromatography is a useful technique for the initial isolation of the different collagen types in their native configuration. Small quantities of collagen mixtures can be rapidly separated into different collagen types with a relatively high degree of purity, based upon stained protein patterns on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) slab gels. In the commonly used bulk salt preparative method for isolating the different collagens, 50 mg of starting material was needed. Three days were required to complete the procedure. The stained protein patterns on SDS-PAGE slab gels showed about 25% contamination with the bulk purified Type III fraction and 20% contamination with the bulk purified type AB collagen. With Zone Precipitation Chromatography 5 mg of starting material was used and in less than 4 hours the mixture was separated with Types III and AB fractions showing less than 10% contamination from other collagen types. The technique is patterned after the Zone Precipitation method reported by Porath seventeen years ago and utilizes a step-wise sodium chloride gradient to precipitate and redissolve the collagens, eluting from the interbead spaces of a molecular sieve column.