Authors: Robert Liddington & Andrey Bobkov
Introduction
Protocol describes expression and purification of vinculin head domain 1.
Reagents
- 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
Procedure
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 2×800ml 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.
Author information
Robert Liddington & Andrey Bobkov, The Burnham Institute
Source: Protocol Exchange (2009) doi:10.1038/nprot.2009.43. Originally published online 5 February 2009.