Preparative Purification using HiPrep™ IMAC FF 16/10

Extracted from Affinity Chromatography Vol. 2: Tagged Proteins, GE Healthcare, 2016

HiPrep™ IMAC FF 16/10 is a ready-to-use 20 ml column, prepacked with uncharged IMAC Sepharose® 6  Fast Flow. The column is well-suited for preparative purification of histidine-tagged recombinant proteins and untagged, naturally occurring proteins. HiPrep™ IMAC FF 16/10 provides fast, simple, and easy separations in a convenient format, and the IMAC Sepharose® 6 Fast Flow medium is well-suited for scaling up.

Prepacked 20 ml HiPrep™  IMAC FF 16/10 column

Fig 3.52. Prepacked 20 ml HiPrep  IMAC FF 16/10 columns allow for easy scale-up.

 

The column is made of polypropylene, which is biocompatible and noninteractive with biomolecules. Separations can be easily achieved using a chromatography system such as ÄKTA. Refer to Chapter 2 (Manual and automated purification), Table 2.1 for a selection guide to purification equipment and to Appendix 1 (Characteristics of Ni Sepharose, Ni Sepharose® excel, TALON® Superflow, and uncharged IMAC Sepharose® products) for a list of HiPrep™ IMAC FF 16/10 column parameters.

IMAC Sepharose® 6 Fast Flow is also available as prepacked 1 ml and 5 ml HiTrap IMAC FF columns and as a bulk medium in lab packs (25 and 100 ml) for packing columns.

 

Sample and buffer preparation

Refer to Purification using IMAC Sepharose® 6 Fast Flow for a general procedure for sample and buffer preparation.

 

Charging the column with metal ion

  1. Prepare a 0.1 M solution of the desired metal ion (e. g., Cu2+, Zn2+, Co2+, Fe3+, or Ni2+) in distilled water. Salts of chlorides, sulfates, etc., can be used (e.g., 0.1 M CuSO4 or 0.1 M NiSO4).

Solutions of Zn2+ ions should have a pH of approximately 5.5 or lower to avoid solubility problems that arise at pH 6 or higher. Fe3+ ions should be immobilized at low pH (approximately pH 3.0) to avoid formation of insoluble ferric compounds.

  1. Wash the column with at least 2 column volumes of distilled water.
  2. Apply at least 0.2 column volumes of the metal ion solution to the column.
  3. Wash the column with at least 5 column volumes of distilled water to remove excess metal ions.

Washing with buffer before applying the metal ion solution may cause unwanted precipitation.


Purification

  1. Apply the centrifuged and/or filtered sample (in binding buffer) to the column at a flow rate of 1 to 10 ml/min (30 to 300 cm/h).
  2. Wash the column with 5 to 10 column volumes of binding buffer at 2 to 10 ml/min (60 to 300 cm/h).
  3. Elute the bound protein with 5 to 10 column volumes of elution buffer at a flow rate of 2 to 10 ml/min (60 to 300 cm/h).
  4. After elution, regenerate the column by washing it with 5 to 10 column volumes of binding buffer. The column is now ready for a new purification.

The column does not need to be stripped and recharged between each purification if the same protein is going to be purified. Reuse of any purification column depends on the nature of the sample and should only be performed with identical tagged proteins to prevent cross-contamination. For more information on this topic and on cleaning and storage, refer to Appendix 1 (Characteristics of Ni Sepharose, Ni Sepharose® excel, TALON® Superflow, and uncharged IMAC Sepharose® products).

IMAC Sepharose® 6 Fast Flow is compatible with reducing agents. However, we recommend removal of any weakly bound metal ions before applying buffer/sample that includes reducing agents. This can be accomplished by performing a blank run without reducing agents (see below). Do not leave or store HiPrep™ IMAC FF 16/10 columns with buffers that include reducing agents.

For critical applications, leakage of metal ions during purification can be diminished by performing a blank run (as described below) before loading sample.

For A280 measurements, use the elution buffers as blanks. If imidazole needs to be removed, use a desalting column (see Chapter 11, Desalting/buffer exchange and concentration). Low-quality imidazole will give a significant background absorbance at 280 nm.

 

Blank run:

Use binding buffer and elution buffer without reducing agents.

  1. Wash the column with 5 column volumes of distilled water (to remove the 20% ethanol).
  2. Wash with 5 column volumes of elution buffer.
  3. Equilibrate with 10 column volumes of binding buffer.

Materials

     
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