Tensin SH2-PTB (ScPc) expression and purification
Methodology describing the expression and purification of the SH2-PTB domain portion of tensin.
30.3KD w/o GST-tag
pI = 8.5
Ext = 0.559
50mM Tris-HCl pH7.0 (at 25°C)
10mM Tris-HCl pH 7.4
1) 100ml o/n culture in LB/amp.
2) Seed 1L TB/amp with 50ml o/n culture.
3) Grow at 37°C until OD600 = 0.6-1.2.
4) Induce with 1mM IPTG.
5) Grow for 3hr, 37°C.
6) Harvest (8krpm, 4°C, 10min). Resuspend pellet in 20ml PBS.
7) LN2 snap freeze.
1) To 1L cell pellet add 1 crushed PI tablet.
2) Lyse cells with 0.2mg/ml lysozyme, 15min rocking or until very thick.
3) Add 10mM MgCl2.
4) Clear DNA with 40µg/ml DNaseI, 15min rocking or until runny.
5) Add 0.1% TX-100. Rock 15min.
6) Spin, 16krpm, 30min, 4°C.
7) Wash 2ml Glutathione Sepharose 4B with 50ml PBS.
8) Rock beads with supernatant for 1hr, 4°C.
9) Load onto a column and collect flow through.
10) Wash beads with 50ml PBS.
11) Wash beads with 50ml Cleavage Buffer.
12) Cleave protein off the beads with 100µl PreScission Protease in 2ml Cleavage Buffer. Rock overnight (16hr, 4°C).
13) Elute the next day with Cleavage Buffer.
14) Dialize into Dialysis buffer. Do protein concentration. Snap Freeze.
15) SDS PAGE.
16) Can further purify on Superdex 75 (10/300) to get rid of upper MW band (do 500µl at a time).
Chill cleavage buffer to 4°C before using.
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Posted 05 Feb, 2009
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