His-tag protein purification

Warning

This protocol was attempted for Tn5 purification. This was unsuccessful, though possibly not because of this protocol.

Source

The QIAexpressionist is the go-to manual on His-tag purifying proteins.

Required solutions

• Lysis buffer: You have options! Check page 113 of the QIAexpressionist.

• 10% PEI: pH to 7.2

Note

Do not use the cOmplete running and elution buffers with normal Ni-NTA His-tag resin! They contain chelating agents that will remove the nickel ions! cOmplete His-tag resin is resistant to small concentrations of chelating agents.

• cOmplete running buffer (native):

  Component	Concentration	g/L final volume
  DI water	90%
  HEPES	20mM	4.766 g
  NaCl	800 mM	46.762 g
  Imidazole	20 mM	1.362 g
  EDTA	1 mM	0.292 g
  DTT	2 mM	0.3085 g
  Glycerol	10%
  NaOH	to pH 7.2

Note

Perform the pH after all components have been added.

• cOmplete elution buffer (native):

  Component	Concentration	g/L final volume	Purpose
  DI water	90%
  HEPES	20mM	4.766 g	Main buffer component
  NaCl	800 mM	46.762 g	High salt maintains protein solubility
  Imidazole	300 mM	20.42 g	Prevents non-specific binding to column.
  EDTA	1 mM	0.292 g	Chelating agent, deactivates proteases and other enzymes.
  DTT	2 mM	0.3085 g	Reducing agent, prevents nonspecific cystine crosslinking.
  Glycerol	10%		Prevents hydrophobic protein-protein interactions.
  NaOH	to pH 7.2

Note

Perform the pH after all components have been added.

• NI-NTA running buffer (native):

  Component	Concentration	g/L final volume	Purpose
  DI water	90%
  NaH2PO4	50mM	6.90 g	Main buffer component.
  NaCl	800 mM	46.762 g	High salt maintains protein solubility.
  Imidazole	15 mM	1.022 g	Prevents non-specific binding to column.
  beta-mercaptoethanol	5 mM		Weaker reducing agent, prevents nonspecific cystine crosslinking.
  Glycerol	10%		Prevents hydrophobic protein-protein interactions.
  NaOH	to pH 8.0

Note

Perform the pH after all components have been added.

Protocol

1. Resuspend the cell pellet in lysis buffer, on ice. For every gram of cell pellet, use 10 mL lysis buffer.

2. Sonicate the resuspended cells, using 5 cycles of 30-second, medium-amplitude 50% duty-cycle sonication.

3. Spin to clarify the lysate using maximum centrifuge speed (14600 xg) for 30 minutes.

4. If the protein of interest has DNA-binding activity, add 1.2 mL/10mL 10% PEI dropwise while stirring. Centrifuge at maximum speed for 30 minutes to remove bound DNA.

5. Fill a column with roughly 1.0 mL resin per gram of cell lysate.

Note

When using affinity columns, the volumes required will be listed as Column Volumes (CVs). The amount of resin is the CV. For a gram preparation, this would mean the CV is 1 mL.

5. Equilibrate the column with 3 CVs of running buffer.

6. Flow the clarified cell lysate across the column, collecting the flow-through.

7. Rinse the column with 8 CVs of running buffer, collecting flow-through fractions.

8. Elute with four separate 0.5 CV washes of elution buffer.

9. Run all collected samples on a SDS-page gel to confirm successful purification.