Keating:Experimental Protocols:Capillary Electrophoresis

Protocol for Purifying TEV

modified NZ/GK 2/05 italics

bold – Dev's changes 8/04

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.

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.

After 4 hours of induction,collect cells by centrifugation (20' at 6000rpm). Save about 500ul before centrifugation to run on a gel.

Resuspend cells in 30 ml of 20 mM tris (pH 8) + .5 M NaCl + No Imidazole in a 50ml conical.

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.

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.

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.

Filter the supernatant.

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. Load sample onto column. Wash with 15 column volumes of same buffer as above. Collect flow through. Save ~100ul for a gel.

Elute with 4 column volumes of 20 mM tris (pH 8) + .5 M NaCl + 120 mM No Imidazol. Save ~100ul for a gel.

Elute with 3 column volumes of 20 mM tris (pH 8) + .5 M NaCl + 300 mM Imidazole.

Add 1mM EDTA and 1mM DTT to all elution fractions.

Run SDS-PAGE gel, and pool fractions accordingly. The gel should be run the same day, as TEV tends to crash out.

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.

Concentrate sample to 1mg/mL using the centricon system.