An independent study shows that Ni-NTA loses less nickel than any other tested resin. Fifty column volumes of buffer containing various additives was passed through a small column containing 100 μl resin from QIAGEN, Supplier G, S, or I. The flowthrough was pooled and a sample sent for analysis by inductively coupled plasma mass spectrometry (ICP-MS) at Dr.Weßling Laboratories, Bochum, Germany according to DIN EN ISO 17025. Native buffer: 50 mM Na phosphate; 300 mM NaCl; 10 mM imidazole, pH 8.0. Denaturing buffer: 100 mM Na phosphate; 10 mM Tris·Cl; 8 M urea.
His-tagged pGAPase was purified in 10 sequential purification procedures. Column load was 10 ml aliquots of cleared E. coli cell lysate containing 30 mg spiked protein. Between purification runs the column was cleaned in place using 0.5 M NaOH. Groups of three samples show column load, flow-through, and peak elution fraction. M: markers.
The indicated protein was purified in buffers containing [A] reducing agent (10 mM DTT) and [B] detergent (1% n-dodecyl-β-D-maltoside) [C] Human GBP1 expressed in S. cerevisiae (data kindly provided by Julia Fres, Center for Molecular Medicine, Cologne University, Germany). M:markers; C: cleared lysate; F: flow-through; W: wash; L: lysate; S: soluble fraction; P: pellet; E: elution fractions.
Ni-NTA outperforms other resins to deliver high yields of high-purity protein. ERK2 was purified using a Ni-NTA Superflow Cartridge or the indicated resin from another supplier, according to manufacturer's instructions. M: markers; C: cleared lysate; F: flow-through; W: wash; E: elution fractions.
See table 'Micro to large-scale purification of IL-1β for a biopharmaceutical project.'
