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**
  - 20mM Tris-HCl
  - 150mM NaCl

### Procedure

**Amplification**

1. 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**

1. 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](http://www.nature.com/protocolexchange/protocols/418) (2009) doi:10.1038/nprot.2009.43. Originally published online 5 February 2009*.