Authors: Robert Liddington & Andrey Bobkov
Introduction
Protocol describes expression and purification of vinculin tail domain from bacterial culture.
Reagents
- 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
Procedure
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 2×800 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
Author information
Robert Liddington & Andrey Bobkov, The Burnham Institute
Source: Protocol Exchange (2009) doi:10.1038/nprot.2009.44. Originally published online 5 February 2009.