( A. Bergh M. Carlsson A...)New gravity-flow column gives faster and simpler purification of histidine-tagged proteins

A. Bergh, M. Carlsson, A. Karlsson, and H. Lindgren
GE Healthcare, Uppsala, Sweden

Histidine-tagged proteins* can be quickly and simply purified on His GraviTrap columns prepacked with Ni Sepharose 6 Fast Flow without the need for a pump or purification system. The protein binding capacity (approx. 40 mg/column) is among the highest available on the market, and separations can be completed in as little as 30 min. Large volumes of clarified or unclarified samples can easily be applied and the purified protein can be eluted in a small volume resulting in a highly concentrated target protein.

Introduction
Immobilized metal ion affinity chromatography (IMAC) is a widely used and highly effective technique that exploits the affinity of histidine-tagged proteins to metal ions attached to chelating separation media, such as Ni Sepharose 6 Fast Flow. His GraviTrap is a single-use column prepacked with Ni Sepharose 6 Fast Flow. His GraviTrap provides high protein binding capacity, short purification times, and simple operation.

The purification of histidine-tagged proteins on His GraviTrap can be divided into four stages: equilibration, sample application, washing, and elution (Fig. 1). Purifications can be performed either under native or denaturing conditions, and a number of additives can be used. In addition, new accessory products further increase speed and convenience.

His GraviTrap and accessories
His GraviTrap contains 1 ml of Ni Sepharose 6 Fast Flow, which delivers a protein binding capacity of approximately 40 mg histidine-tagged protein/column. The columns are made of bio-inert polypropylene, and special frits protect the medium from running dry during use. The column packaging can be converted into a stand (Workmate), and the plastic tray in this package can b
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