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New gravity-flow column gives faster and simpler purification of histidine-tagged proteins

roximately four times faster on His GraviTrap (Fig 2B).

Complete details of these comparisons are provided in data file 11-0036-90, available at www.amershambiosciences.com/promo_dm205

His GraviTrap also enables rapid separation of large proteins. To demonstrate this, His GraviTrap was used to purify (His)10-TRX-P450 (Mr 133 200). E. coli JM109 cells expressing (His)10-TRX-P450 were enzymatically and mechanically lysed and a 20 ml clarified sample was loaded onto His GraviTrap using LabMate. The whole purification took just 25 min.

Results show that the purification yielded three major protein bands when analyzed by SDS-PAGE (Fig 3A, lane 4). Western blot analysis confirms that each of the three bands contains a histidine-tag (Fig 3B). However, only a weak band can be seen from the band at approximately Mr 15 000. Some of the smaller proteins may have passed through the membrane due to a prolonged blotting time. N-terminal sequencing (data not shown) of the three bands confirms that the low molecular weight bands are truncated forms of the histidine-tagged target protein. To separate the full-length protein from the truncated forms, a second purification step, such as gel filtration, is recommended.


Summary
His GraviTrap columns provide a very fast and simple method for purifying histidine-tagged proteins without the need for a pump or purification system. The prepacked Ni Sepharose 6 Fast Flow gives high protein binding capacity. Proven protocols, application data, and new support products help optimize purification results and simplify the whole procedure.

To obtain data file 11-0036-90, visit www.am
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