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162 Godat et al.
3.7.1. Purification of HQ-Tagged Proteins Expressed in Wheat Germ
The TNT® SP6 High-Yield Protein Expression System is a single-tube,
coupled transcription/translation system which can express up to 100 μg/ml of
protein. This cell-free expression system contains all the components (tRNA,
ribosomes, amino acids, polymerase and translation initiation, elongation
and termination factors) necessary for protein synthesis directly from DNA
templates. In general, wheat germ extracts provide some co-translational and
post-translational modifications such as phosphorylation (15), farneslylation
(16) and myristoylation (17).
1. Add 150 μl of MagneHis Bind/Wash buffer + 500 mM NaCl to 50 μl wheat
germ reaction.
2. Vortex the MagneHis Ni Particles to a uniform suspension.
3. Add 30 μl of MagneHis Resin to the reaction. Mix and incubate for 5 min. Mix
periodically to keep the particles from settling. Mix by pipetting or flicking tube.
4. Place in magnetic stand and remove supernatant.
5. Add 150 μl of MagneHis Bind/Wash buffer + 500 mM NaCl. Mix and place in
magnetic stand (see Note 9).
6. Repeat step 5 two more times for a total of three washes.
7. Add 100 μl of MagneHis Elution buffer and mix. Incubate 1–2 min and then
place in magnetic stand. Supernatant will contain purified protein.
3.7.2. Purification of HQ-Tagged Proteins Expressed in Rabbit
Reticulocyte Lysate
The TNT® Quick Coupled Transcription/Translation System is a singletube,
coupled transcription/translation reaction that contains RNA polymerase,
nucleotides, salts and recombinant Rnasin®, ribonuclease, inhibitor, for
eukaryotic in vitro translation. Canine microsomal membranes may be added
for post-translational modifications such as signal sequence cleavage and glycosylation.
1. Add 150 μl of MagZ Bind/Wash buffer to 50 μl rabbit reticulocyte reaction.
2. Vortex the MagZ Particles to a uniform suspension and add 60 μl of MagZ
Resin to 1.5 ml tube. Place in magnetic stand and remove buffer.
3. Add rabbit reticulocyte reaction diluted in buffer to resin. Mix and incubate for
15 min. Mix periodically to keep the particles from settling. Mix by pipetting or
flicking tube.
4. Place in magnetic stand and remove supernatant.
5. Add 150 μl of MagZ Bind/Wash buffer. Mix and place in magnetic stand.
6. Repeat step 5 three more times for a total of four washes.
7. Add 100 μl of MagZ Elution Buffer and mix. Incubate 1–2 min at room
temperature and then place in magnetic stand. Supernatant will contain purified
protein.