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152 Godat et al.
tag. HQ-tagged proteins are not only expressed and purified similarly to a
polyhistidine-tagged protein, but can also be purified from bacterial cells under
native and denaturing conditions. The characteristics of the HQ tag are (i) small
size, (ii) can be purified using IMAC methods, (iii) many HQ-tagged proteins
eluted from metal affinity resin at a low imidazole concentration (e.g., 50 mM
imidazole) and (iv) the HQ tag can be attached at amino-(N) or carboxy-(C)
termini of the proteins.
2. Materials
2.1. Small-Scale Magnetic Nickel Purification for Bacteria
1. MagneHis Protein Purification System (cat. no. V8500, Promega)—MagneHis
Binding/Wash Buffer: 100 mM HEPES + 10 mM imidazole, pH 7.5; MagneHis
Elution Buffer: 100 mM HEPES (4-2-Hydroxyethyl) piperazine-1-elthanesulfonic
acid) + 500 mM imidazole, pH 7.5; MagneHis Ni Particles; FastBreak Cell
Lysis Reagent, 10×; DNase I.
2. Magnetic stand (cat. no. Z5342, Promega).
3. NaCl (5 M).
2.2. Small-Scale Non-Magnetic Nickel Purification for Bacteria
1. HisLink Spin Purification System (cat. no. V1320, Promega)—HisLink
Binding/Wash Buffer: 100 mM HEPES + 10 mM imidazole, pH 7.5; HisLink
Elution Buffer: 100 mM HEPES + 500 mM imidazole, pH 7.5; HisLink Protein
Purification Resin; HisLink Spin Columns; FastBreak Cell Lysis Reagent,
10×; DNase I; collection tubes.
2. Microcentrifuge.
3. Vacuum Manifold (cat. no. A7231, Promega).
4. Vacuum Adapter (cat. no. A1331, Promega).
5. NaCl (5 M).
2.3. Large-Scale Non-Magnetic Nickel Purification for Bacteria
1. HisLink Resin (cat. no. V8821, Promega).
2. Columns.
3. Binding buffer: 100 mM HEPES + 10 mM imidazole, pH 7.5.
4. Wash buffer: 100 mM HEPES + 10–20 mM imidazole, pH 7.5.
5. Elution buffer: 100 mM HEPES + 50–1000 mM imidazole, pH 7.5.
6. NaCl (5 M).
2.4. Magnetic Nickel Purification for Mammalian and Insect Cells
1. MagneHis Protein Purification System (cat. no. V8500, Promega)—MagneHis
Binding/Wash Buffer: 100 mM HEPES + 10 mM imidazole, pH 7.5; MagneHis