Inoculate 2ml of 5ml o/n culture of either p3133 (empty vector pET11a) or p3134 (dnEBNA-1/Soft) in E. coli BL21 LysS per 0.5L LB + ampicillin (grow two 0.5L cultures of each)

Incubate ~2hrs @37C 250rpm until OD600 = 0.4-0.6

Induce with 5ml 100mM ITPG per 0.5L culture

Incubate 2-3hrs @37C 250rpm

Spin down 2 flasks of each (1L total) in 500ml GSA centrifuge bottles 5000rpm, 4C, 10min

Decant supernatant, freeze pellet @-20C

Resuspend pellet in TED+0.15M NaCl (2-5ml per gram wet weight of pellet; for bacterial pellet from 1L I used 6ml on 02/2004)

Add 200x lysozyme and 100x proteasome inhibitors to 1x final concentration

Transfer to disposable, sterile tube and incubate on ice 30min (omitted on 02/2004)

Sonicate setting 10, 15-30sec bursts, 6 rounds, incubate on ice ~2min between rounds

Transfer to microfuge tubes; Centrifuge 4C, 10k rpm 30min

Transfer supernatant to fresh tube.

Column Preparation

a. Resuspend matrix as 50% slurry and load closed column

b. Wash twice with 5 bed volumes of TED+0.15M NaCl

c. Open column and drain to form compact 100% slurry

Load sample onto column. Note: all flow rates should be 0.5ml/min

Collect Flow Through (FT), pass FT over column again. Save 50ul of supernatant as FT.

Wash with 25ml TE+0.15M NaCl. Collect this wash and save 50ul as Wash 1.

Wash with 15ml TE+0.5M NaCl, collect in 5ml fractions. Save 50ul of each as Wash 2.1, Wash 2.2, Wash 2.3.

Elute dnEBNA-1/Soft from column with 5-10ml TE+0.7M NaCl+30% propylene glycol, collect in 1ml fractions. Add 5ul of 0.02M DTT to each 1ml fraction. Save 50ul of each fraction as Elution 1, 2, 3, 4, 5 (6, 7, 8, 9, 10).

Run 2 identical SDS-PAGE gels with all bold samples above, protein size marker, and concentration standards (BSA 0.1ug, 1ug, 10ug) or protein positive for Soft-tag and/or EBNA-1

d. Run one gel as a Coomassie Blue stain for bulk protein levels

e. Run one gel as Western for identification of Soft tag and/or EBNA-1

TED+0.15M NaCl

10mM Tris pH 7.4

1mM EDTA (pH 8)

150mM NaCl

0.1mM DTT

TE+0.5M NaCl

10mM Tris pH 7.4

1mM EDTA (pH 8)

500mM NaCl

10% glycerol (v/v)

TE+0.7M NaCl+30% propylene glycol

10mM Tris pH 7.4

1mM EDTA (pH 8)

750mM NaCl

30% propylene glycol (v/v)