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156 Godat et al.
4. Invert tube to mix (∼10 times) and incubate for 2 min at room temperature.
Make sure the MagneHis Ni Particles are well mixed.
5. Place the tube in the appropriate magnetic stand for approximately 30 s to capture
the MagneHis Ni Particles. Using a pipette carefully remove the supernatant.
6. Remove the tube from the magnetic stand. Add 150 μl of MagneHis
Binding/Wash Buffer to the MagneHis Ni Particles and pipette to mix. If NaCl
was added for binding, also use the same amount of NaCl during washing. Make
sure that particles are resuspended well.
7. Place the tube in the magnetic stand for approximately 30 s to capture the
MagneHis Ni Particles. Using a pipette, carefully remove the supernatant.
8. Repeat the wash step two times for a total of three washes.
9. Remove the tube from the magnetic stand. Add 100 μl of MagneHis Elution
Buffer and pipette to mix.
10. Incubate for 1–2 min at room temperature. Place in a magnetic stand to capture
the MagneHis Ni Particles. Using a pipette, remove the supernatant containing
the purified protein.
3.3. Lysis and Purification from Bacterial Culture Using
Non-Magnetic Ni Particles (Spin Baskets)
Lysis of bacterial cells and binding of HQ-tagged proteins is done in one
step. Purification can be done by centrifugation or using a vacuum manifold.
3.3.1. Direct Lysis of Bacterial Cell Cultures Using Lysis Buffer
Cultures with concentrations up to 8 OD 600 nm units/ml have been successfully
used with the HisLinkSpin Protein Purification System. A maximum
of 700μl of bacterial culture can be loaded per HisLink Spin Column.
1. Pipette 700 μl of bacterial culture into a 1.5 ml microcentrifuge tube. Add 70 μl
of the FastBreak Reagent/DNase I solution (see Note 5).
2. Resuspend the resin and allow it to settle. Once the resin has settled, use a widebore
pipette tip to transfer 75 μl of the HisLink Resin from the settled resin bed
to the 1.5 ml microcentrifuge tube.
3. Adding 200 mM NaCl prior to the addition of the HisLink Resin may reduce
non-specific binding and improve binding of HQ-tagged proteins. If NaCl is used
in binding also use NaCl in the washes.
4. Incubate the sample and resin for 30 min, mixing frequently on a rotating platform
or shaker to optimize binding.
5. Continue with either the centrifugation or vacuum spin column protocol.
3.3.2. Centrifugation Protocol for Spin Columns
1. Place a HisLink Spin Column onto a collection tube (or a new 1.5 ml microcentrifuge
tube). Use a wide-bore pipette tip to transfer the lysate and resin from
the original 1.5 ml microcentrifuge tube to the spin column.