Methodology describing the expression and purification of the SH2-PTB domain portion of tensin.
Method Article
Tensin SH2-PTB (ScPc) expression and purification
https://doi.org/10.1038/nprot.2009.20
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
You are reading this latest protocol version
tensin SH2-PTB domain
expression
purification
Methodology describing the expression and purification of the SH2-PTB domain portion of tensin.
pGEX4T1-GST-ScPc
30.3KD w/o GST-tag
pI = 8.5
Ext = 0.559
Cleavage Buffer
50mM Tris-HCl pH7.0 (at 25°C)
150mM NaCl
1mM EDTA
1mM DTT
Dialysis Buffer
10mM Tris-HCl pH 7.4
50mM NaCl
0.5mM EGTA
1mM DTT
Expression
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.
Purification
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.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
You are reading this latest protocol version