GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden
Sponsored by GE Healthcare Published in July 2011 (p.16) Sponsored,vendor-submitted protocol
HisTrap FF crude is prepacked with Ni Sepharose™ 6 Fast Flow, which is intended for immobilized metal affinity chromatography (IMAC) and especially histidine-tagged protein purification. The chromatography medium has low Ni2+-leakage and is compatible with a wide range of buffers and additives, including denaturants, detergents and reducing agents.
In this study, several histidine-tagged proteins from sonicated E. coli crude cell lysates were purified to establish recovery and purity of the target protein and capacity of the column. A number of different mechanical lysis techniques, such as sonication, freeze/thaw and homogenization, resulted in similar purification performance on the HisTrap FF crude column.
Comparative experiments performed with the equivalent HisTrap FF column, using clarified sample as a reference, showed no difference in target protein pool volume, recovery and purity, but saved sample preparation time as no centrifugation and filtration of the sample before loading on the column was necessary. Sample preparation using HisTrap FF crude columns is simply performed by enzymatic and mechanical lysis (the mechanical lysis was somewhat extended to ensure complete lysis). In, for example, a screening situation with a number of samples, the HisTrap FF crude column would reduce the total purification time considerably and minimize the possible degradation of the target protein.