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| == Protocol for Purifying TEV ==
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| modified NZ/GK 2/05 ''italics''
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| '''bold''' – Dev's changes 8/04
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| Innoculate a single colony containing the TEV plasmids (pRK793 and pRIL in BL21(DE3) cells) in 5mL of LB containing 100ug/mL ampicillin and 35ug/mL chloramphenicol. Incubate on the roller wheel in the warm room overnight.
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| Innoculate 1L LB containing 100ug/mL ampicillin and 35ug/mL chloramphenicol with 1mL of overnight culture. Grow at 37 degrees on the shaker until mid log phase (OD600 ~ 0.5). Save about 500ul to run on a gel. Induce cells with IPTG to give a final concentration of 1mM and reduce the temperature to 30 degrees and return to the shaker.
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| After 4 hours of induction,collect cells by centrifugation (20' at 6000rpm). Save about 500ul before centrifugation to run on a gel.
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| Resuspend cells in '''30 ml of 20 mM tris (pH 8) + .5 M NaCl + No Imidazole''' ''in a 50ml conical''.
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| Lyse cells using a sonicator. For a 1 L bacterial prep, sonicate on hold for 10 seconds, then set on ice for 10 seconds. Repeat 8 to 10 times.
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| Determine the lysate volume. Add 5% polytheleneimine (pH 7.9 adjusted with Hcl) to a final concentration of 0.1%. ''For 30ml, add 1.5ml''.
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| Mix the lysate by inversion, ''transfer to a centrifuge tube'', and centrifuge at 14,000rpm for 30min. Save a small portion of supernatant and pellet to run on a gel. The supernatant is what you want to save and run on a column.
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| Filter the supernatant.
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| Drain off the NiNTA EtOH storage solution, then equilibriate a NiNTA column. Use 4 column volumes of '''same buffer as above'''. Use 1mL packed resin for every 8mg of protein. Estimate how much protein by running on a gel and comparing to marker, ''or ~3ml resin / 1L culture.
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| ''
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| Load sample onto column. Wash with 15 column volumes of '''same buffer as above'''. Collect flow through. Save ~100ul for a gel.
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| Elute with 4 column volumes of '''20 mM tris (pH 8) + .5 M NaCl + 120 mM No Imidazol'''. Save ~100ul for a gel.
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| Elute with 3 column volumes of '''20 mM tris (pH 8) + .5 M NaCl + 300 mM Imidazole'''.
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| ''Add 1mM EDTA and 1mM DTT to all elution fractions''.
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| Run SDS-PAGE gel, and pool fractions accordingly. The gel should be run the same day, as TEV tends to crash out.
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| Load onto S-200 column in cold room and run using the following buffer: 25mM PO4 (pH 8.0), 200mM NaCl, 10% glycerol, 2mM EDTA, and 10mM DTT. Pool fractions.
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| Concentrate sample to 1mg/mL using the centricon system.
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