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

e used to collect liquid waste.

The optional LabMate reservoir increases the loading capacity from 10 ml to approximately 35 ml. Large volumes can thus be applied in one go without having to constantly attend the column to add more sample.

The new His Buffer Kit delivers added convenience by providing phosphate buffer concentrates and highly pure 2 M imidazole stock solutions. This kit eliminates time-consuming manual buffer preparation, thereby promoting optimal convenience, performance, and reproducibility.

Faster purifications under a wide range of conditions
The performance of His GraviTrap was compared with Ni-NTA Superflow™ gravity-flow column (Qiagen Inc) during the purification of histidine-tagged green fluorescent protein (GFP-[His]6; Mr 28 000) from an E. coli lysate. The imidazole concentration in the sample and binding buffers was 45 mM for His GraviTrap and 10 mM for Ni-NTA Superflow (the latter according to manufacturer’s recommendations).

The recovery was greater than 98% in the first 3 ml eluate from His GraviTrap, and approximately 80% from the other column. For complete elution from Ni-NTA Superflow, a total volume of 6 to 9 ml was needed, and the total purification time was close to 100 min. The total purification time was 20 min when His GraviTrap was used (Fig 2A). The purity of the eluates was similar as determined by SDS-PAGE (data not shown).

His GraviTrap and Ni-NTA Superflow columns were also compared under denaturing conditions. Histidine-tagged Maltose Binding Protein (MBP-(His)6; Mr 43 000) in E. coli BL-21 lysate containing 7 M urea was applied to the columns. Elution was achieved by lowering the pH from pH 8.0 to pH 4.5. Although denaturing conditions increase the purification time due to the higher viscosity of the samples and buffers, total purification time was still app
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