Isolation Purification and Separation

scientificprotocols authored almost 3 years ago

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

  1. 100ml o/n LB/amp culture per 800ml culture the next day, 37°C.
  2. 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.
  3. Spin 8krpm, 15min, 4°C.
  4. Resuspend in His Binding Buffer. Snap freeze.

Purification

  1. Lyse 2×800 ml cell pellets with homogenizer.
  2. Spin at 16 krpm, 30min, 4°C.
  3. Load supernatant on to 3 ml equilibrated Ni-NTA column.
  4. Wash with 50 ml binding buffer.
  5. Wash with 10 column volumes wash buffer.
  6. Elute with elution buffer.
  7. Dialyze protein into dialysis buffer 1.
  8. Concentrate VD1 to 20-30 mg/ml.
  9. Dialyze protein into dialysis buffer 2 or end use buffer.
  10. SDS PAGE on purification.
  11. 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.

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