Purification of dnEBNA-1/Soft from E. coli BL21 LysS

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 37℃250rpm until OD600 = 0.4-0.6

Induce with 5ml 100mM ITPG per 0.5L culture

Incubate 2-3hrs 37℃250rpm

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

Decant supernatant,freeze pellet @20℃

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; Centrifuge4℃ 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 50μl of supernatant as FT.

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

Wash with 15ml TE+0.5M NaCl,collect in 5ml fractions.Save 50μl 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 5μl of 0.02M DTT to each 1ml fraction.Save 50μl 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 0.1μg,1μg,10μg)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)