Protocol describes expression and purification of vinculin head domain 1.
Method Article
VINCULIN HEAD Domain 1 (Vd1 and Vd1 Y100E mutant): expression and purification
https://doi.org/10.1038/nprot.2009.43
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vinculin
Protocol describes expression and purification of vinculin head domain 1.
Vector - pET15b-VD1
Molecular Weight = 30.8 KD
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
pH 8.0
20mM Tris-HCl pH 7.9
Elution Buffer
75mM Imidazole
500mM NaCl
pH 8.0
20mM Tris-HCl pH 7.9
Dialysis Buffer 1
25mM Tris-HCl pH 8.0
150mM NaCl
5mM EDTA
5mM βME
Dialysis Buffer 2
25mM Tris-HCl
150mM NaCl
5mM βME
ITC Buffer
20mM Tris-HCl
150mM NaCl
Amplification
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 2x800ml cell pellets with homogenizer.
Spin 16krpm, 30min, 4 °C.
Load sup on to 3ml equilibrated Ni-NTA column.
Wash with 50ml binding buffer.
Wash with 10 column volumes wash buffer.
Elute with elution buffer.
Dialyze protein into dialysis buffer 1.
Concentrate VD1 to 20-30mg/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
You are reading this latest protocol version