Ni-NTA Spin Kit (50)

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Protocol tips
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Publication protocol

We divided our QIAcube protocol into 4 parts (A, B, C and D) to allow for the rotor adapters to be emptied, tips to be refilled, samples to be taken or buffers to be changed between the steps. A summary of the purification process can be seen in Table 1. During part A the column with 300 µl of Ni-NTA beads in 20% ethanol is equilibrated with lysis buffer and the lysate load is added to the column in several steps with incubation periods to maximize binding. During part B, the column is washed and the first 800 µL elution fraction is collected in elution position 3 of the rotor adapter (Figure 1). During part C, the second 800 µL elution fraction is collected in the middle position 2 of the rotor adapter and the column is washed. During part D, the column is regenerated and stored for future use. The elution fractions are dialyzed in 10 mM Tris, 100 mM NaCl, pH 8 to remove elution buffer components (guanidine or imizadole) and spun at high speed in a microcentrifuge before the concentration is measured. All steps performed by the QIAcube scripts (Protein Resin Part A, B, C and D) are found in Supplementary File A and can be obtained from moc.negaiq@balsnoitacilppa to run on any QIAcube. Purification using the Ni-NTA spin kit (Qiagen) was performed according to the manual using 2 mL cell culture for each purification. All elutions were quantitated using absorbance measurements at 280 nm. Any kD Mini-PROTEAN TGX gels from Bio-Rad (Cat. # 456-9036) were stained using Labsafe Gel blue from G-Biosciences (Cat. # 786-35).

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Manufacturer protocol

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