Authors: Robert Liddington & Andrey Bobkov
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)
- 150mM NaCl
- 1mM EDTA
- 1mM DTT
- 10mM Tris-HCl pH 7.4
- 50mM NaCl
- 0.5mM EGTA
- 1mM DTT
- 100ml o/n culture in LB/amp.
- Seed 1L TB/amp with 50ml o/n culture.
- Grow at 37°C until OD600 = 0.6-1.2.
- Induce with 1mM IPTG.
- Grow for 3hr, 37°C.
- Harvest (8krpm, 4°C, 10min). Resuspend pellet in 20ml PBS.
- LN2 snap freeze.
- To 1L cell pellet add 1 crushed PI tablet.
- Lyse cells with 0.2mg/ml lysozyme, 15min rocking or until very thick.
- Add 10mM MgCl2.
- Clear DNA with 40µg/ml DNaseI, 15min rocking or until runny.
- Add 0.1% TX-100. Rock 15min.
- Spin, 16krpm, 30min, 4°C.
- Wash 2ml Glutathione Sepharose 4B with 50ml PBS.
- Rock beads with supernatant for 1hr, 4°C.
- Load onto a column and collect flow through.
- Wash beads with 50ml PBS.
- Wash beads with 50ml Cleavage Buffer.
- Cleave protein off the beads with 100µl PreScission Protease in 2ml Cleavage Buffer. Rock overnight (16hr, 4°C).
- Elute the next day with Cleavage Buffer.
- Dialize into Dialysis buffer. Do protein concentration. Snap Freeze.
- SDS PAGE.
- 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.
Robert Liddington & Andrey Bobkov, The Burnham Institute
Source: Protocol Exchange (2009) doi:10.1038/nprot.2009.20. Originally published online 5 February 2009.