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Purification of HQ-Tagged Proteins 153
Elution Buffer: 100 mM HEPES + 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).
4. Imidazole (1 M).
2.5. Magnetic Nickel Purification for Cell-Free Expression:
Wheat Germ Extract
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 + 500 mM imidazole, pH 7.5; MagneHis Ni
Particles; FastBreak Cell Lysis Reagent, 10×; DNase I.
2. Magnetic Stand (cat. no. Z5342, Promega).
3. TNT® SP6 High-Yield Protein Expression System (cat. no. L3261, Promega)—
TNT® SP6 High-Yield Master Mix; Nuclease-Free Water.
4. NaCl (5 M).
2.6. Magnetic Purification Purification for Cell-Free Expression:
Rabbit Reticulocyte Lysate
1. MagZ Protein Purification System (cat. no. V8830, Promega)—MagZ
Binding/Wash Buffer: 20 mM sodium phosphate + 500 mM NaCl, pH 7.4; MagZ
Elution Buffer: 1 M imidazole, pH 7.5; MagZ Binding Particles.
2. Magnetic Stand (cat. no. Z5342, Promega).
3. TNT® SP6 Quick Coupled Transcription/Translation System (cat. no. L2080,
Promega)—TNT® Quick Master Mix; SP6 Luciferase Control DNA; Methionine
(1 mM); Luciferase Assay Reagent; Nuclease-Free Water.
2.7. Magnetic Nickel Purification for Automation
1. Maxwell16 Instrument (cat. no. AS1000, Promega)—Instrument; Power Cable;
RS-232 Cable for Firmware Upgrades; 1.5 mm Hex Wrench; Cartridge Preparation
Rack; Magnetic Elution Tube Rack.
2. Maxwell16 Polyhistidine Protein Purification Kit (cat. no. AS1060, Promega)—
Maxwell16 Polyhistidine Protein Purification Sample Cartridges; Elution Buffer:
100 mM HEPES + 500 mM imidazole, pH 7.5.
3. NaCl (5 M).
2.8. Non-Magnetic Nickel Purification for High Throughput
1. Hislink96 Protein Purification System (cat. no. V3680, Promega)—
Binding/Wash Buffer: 100 mM HEPES + 10 mM imidazole, pH 7.5; Elution
Buffer: 100 mM HEPES + 500 mM imidazole, pH 7.5; HisLink Resin;
FastBreak Cell Lysis Reagent, 10×; DNase; Filtration Plate; Collection Plate.