Protocol describes expression and purification of vinculin tail domain from bacterial culture.
Method Article
VINCULIN TAIL (878-1065aa, Wt and Y1064E mutant), expression and purification
https://doi.org/10.1038/nprot.2009.44
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vinculin
Protocol describes expression and purification of vinculin tail domain from bacterial culture.
pET15b-VD1
MW=30.8
Ext = 10870 to get µM
Ext = 0.355 to get µg/µl
Binding Buffer
5mM Imidazole
500mM NaCl
20mM Tris-HCl pH 7.9
Wash Buffer
30mM Imidazole
500mM NaCl
20mM Tris-HCl pH 7.9
Elution Buffer
75mM Imidazole
500mM NaCl
20mM Tris-HCl pH 7.9
Dialysis Buffer 1
25mM Tris-HCl pH 8.0
150mM NaCl
5mM EDTA
5mM bME
Dialysis Buffer 2
25mM Tris-HCl pH 8.0
150mM NaCl
5mM bME
ITC Buffer
20mM Tris-HCl pH 8.0
150mM NaCl
Expression
100ml o/n LB/amp culture per 800ml culture the next day, 37°C.
Seed 800ml LB/amp culture with 100ml o/n culture. Grow at 37°C until OD600 = 0.6-1.2.
Induce with 1mM IPTG. Grow 3hr. Harvest.
Spin 8krpm, 15min, 4°C.
Resuspend in His Binding Buffer. Snap freeze.
Purification
Lyse 2x800 ml cell pellets with homogenizer.
Spin at 16 krpm, 30min, 4°C.
Load supernatant on to 3 ml equilibrated Ni-NTA column.
Wash with 50 ml binding buffer.
Wash with 10 column volumes wash buffer.
Elute with elution buffer.
Dialyze protein into dialysis buffer 1.
Concentrate VD1 to 20-30 mg/ml.
Dialyze protein into dialysis buffer 2 or end use buffer.
SDS PAGE on purification.
Dialyze protein into appropriate experimental buffer
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
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