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Purification of HQ-Tagged Proteins 161
10. Place the tube in the appropriate magnetic stand for approximately 30 s. Allow
the MagneHis Ni particles to be captured and carefully remove the supernatant
using a pipette.
11. Repeat the wash step two times for a total of three washes.
12. Remove the tube from the magnetic stand. Add 100 μl of MagneHis Elution
Buffer and pipette to mix.
13. Incubate for 1–2 min at room temperature. Place the tube in a magnetic stand to
capture the MagneHis Ni Particles with the magnet. Using a pipette, remove
the supernatant containing the purified protein.
3.6.3. Purification of Secreted HQ-Tagged Proteins from Insect
or Mammalian Cell (See Note 8)
1. Vortex the MagneHis Ni Particles to a uniform suspension (see Note 4).
2. Add 30 μl of MagneHis Ni Particles to 1 ml of culture medium after removing
cells.
3. Add 1 M imidazole to a final concentration of 20 mM to decrease non-specific
binding of serum proteins (20 μl/1 ml sample). Adding 500 mM NaCl may improve
HQ-tagged protein binding and decrease non-specific binding.
3. Invert tube to mix (∼10 times) and incubate for 2 min at room temperature.
4. Place the tube in the appropriate magnetic stand for approximately 30 s to capture
the MagneHis Ni Particles with the magnet. Using a pipette, carefully remove
the supernatant.
5. Remove the tube from the magnet. Add 500 μl of MagneHis Binding/Wash
Buffer containing 500 mM NaCl to the MagneHis Ni Particles and pipette to
mix. Make sure that the particles are resuspended well.
6. Place the tube in the appropriate magnetic stand for approximately 30 s to capture
the MagneHis Ni Particles with the magnet. Using a pipette, carefully remove
the supernatant.
7. Repeat the wash step two times for a total of three washes.
8. Remove the tube from the magnet. Add 100 μl of MagneHis Elution Buffer and
pipette to mix.
9. Incubate for 1–2 min at room temperature. Place the tube in a magnetic stand to
capture the MagneHis Ni Particles. Using a pipette, remove the supernatant that
contains the purified protein.
3.7. Purification of HQ-Tagged Proteins Expressed in Cell-Free
Expression Systems
Cell-free expression systems may be preferred over in vivo or native systems,
because they can be used for the expression of toxic, proteolytically sensitive
or unstable proteins (11–13). In vitro systems provide the ability to incorporate
non-natural amino acids containing photoactivatable fluorescent or biotin
residues or radioactive amino acids (14). The HQ can be utilized in cell-free
expression systems.