Preparative Purification Using HisPrep™ FF 16/10

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

HisPrep™ FF 16/10 columns are specially designed 20 ml HiPrep columns, ready to use for easy, one-step preparative purification of histidine-tagged proteins. Prepacked with Ni Sepharose® 6 Fast Flow, the columns exhibit high binding capacity and excellent flow properties. For easy scale-up, columns can be connected in series (back pressure will increase).

HisPrep™ FF 16/10 column

Fig 3.27. HisPrep™ FF 16/10 column for convenient preparative purification of histidine-tagged proteins.

 

The column is made of polypropylene, which is biocompatible and noninteractive with biomolecules. Purifications can be easily achieved using a chromatography system such as ÄKTA or other chromatography systems. 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 HisPrep™ FF 16/10 column parameters. Note that HisPrep™ FF 16/10 columns cannot be opened or refilled.

 

Sample and buffer preparation

Refer to Purification using Ni Sepharose® 6 Fast Flow earlier in this chapter for a general procedure for sample and buffer preparation.

 

Purification

For first-time use, it is important to set an appropriate pressure limit on the system (maximum pressure over the packed bed is 1.5 bar).

  1. Equilibrate with at least 5 column volumes of binding buffer. Avoid introducing air into the column.
  2. 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).
  3. Wash the column with 5 to 10 column volumes of binding buffer at 2 to 10 ml/min (60 to 300 cm/h).
  4. 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).
  5. After elution, regenerate the column by washing it with approximately 100 ml of binding buffer. The column is now ready for a new purification of the same target protein.

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).

Ni Sepharose® 6 Fast Flow is compatible with reducing agents. However, we recommend removal of any weakly bound Ni2+ 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 store HisPrep™ columns with buffers that include reducing agents.

Leakage of Ni2+ from Ni Sepharose® 6 Fast Flow is low under all normal conditions. For very critical applications, leakage during purification can be even further 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:

  1. Use binding buffer and elution buffer without reducing agents.
  2. Wash the column with 5 column volumes of distilled water (to remove the 20% ethanol).
  3. Wash with 5 column volumes of elution buffer.
  4. Equilibrate with 10 column volumes of binding buffer.

 

Application example

Refer to Figure 3.13 (Purification using HisTrap HP and HisTrap FF).

Materials

     
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