His-tag protein purification

Abstract

Affinity purifcation of His-tagged proteins on HisTrap column.

1M Tris-HCl stock
1M Imidazole stock
- 68.08g Imidzaole
- ad 1L
2L Base-buffer for lysis and binding and everything [27.5mM Tris-HCl pH 7.4, 0.55M NaCl, 44mM Imidazole, 11% Glycerol]
- +252.87 ml Glycerol from 87% stock (into glass beaker as the first step)
- +55 mL 1M Tris-HCl
- fill to about 1.7 L with ice-cold water
- +64.28g NaCl
- +88 mL 1M Imidazole
- adjust pH to 7.4
- ad 2L
0.25L Lysis buffer -- for chemical lysis, the idea is to add bugbuster after dissolving the pellet [final: 25mM Tris-HCl pH 7.4, 0.5M NaCl, 40mM Imidazole, 10% Glycerol, 1 mM TCEP, 1xBugbuster, Compete, Benzonase 25U/ml, lysozyme]
- 0.25L Base-Buffer (27.5mM Tris-His...) (use 0.225 for perfectly accurate)
- add before use:
  - 5 tablets of ComPete Protease inhibitor EDTA-free (normal, not the Mini)
  - 2.5 uL Benzonase (380 U/µl)
  - 0.25 mL 1M TCEP
- after dissolving pellet in 36mL buffer, add:
  - 4 mL 10x Bugbuster to 1x
  - spatula of Lysozyme
1L Binding Buffer
- 0.9L base-buffer buffer
- 0.1L H2O
- add before use:
  - 1 mL 1M TCEP
0.25L Elution Buffer [=Binding Buffer with 0.5M Imidazole, 1mM TCEP]
- add 8. g Imidazole to Binding buffer
- add before use: 0.25mL 1M TCEP

thaw pellet by putting tube into cold water
Lysis in 50 mL Falcon tubes
- add 36 mL lysis-base buffer, dissolve by vortexing
- (optimally 5 mL buffer / 1g pellet wet weight)
- add 4 mL 10xBugbuster
- add 1 spatula of Lysozyme
- incubate 20-40 min rotating in cold room
Clarify
- transfer into 2 x 30 mL centrifuge tubes
- spin down 45min @ 80.000g
- filter clarified lysate through Miracloth into 50 mL Falcon or 100 mL glass bottle
Affinity capture (AKTA)