Purification using StrepTactin Sepharose® High Performance

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

StrepTactin is a specially engineered streptavidin ligand. The binding affinity of Strep-tag II to the immobilized ligand is nearly 100-fold higher than to streptavidin, making StrepTactin Sepharose® High Performance ideal for purifying Strep-tag II proteins.

The small bead size (average 34 µm) of the Sepharose® High Performance matrix results in high-resolution separations, sharp peaks, and purified target proteins in a concentrated form.  StrepTactin Sepharose® High Performance is compatible with a wide range of additives, tolerates all commonly used aqueous buffers, and is quickly and easily regenerated using  0.5 M NaOH. See Appendix 4 (Characteristics of StrepTactin Sepharose® High Performance products) for more information on the characteristics of the medium.


Column packing

See instructions supplied with the product or refer to Appendix 6 (Column packing and preparation) for general guidelines for column packing.


Sample preparation

Adjust the sample to the composition of the binding buffer (see below). For example, dilute the sample with binding buffer or buffer exchange using a desalting column (see Chapter 11, Desalting/buffer exchange and concentration).

Pass the sample through a 0.22 µm or a 0.45 µm filter and/or centrifuge it immediately before applying it to the column. If the sample is too viscous, to prevent it from clogging the column dilute it with binding buffer, increase lysis treatment (sonication, homogenization), or add DNase/RNase to reduce the size of nucleic acid fragments.


Buffer preparation

Binding buffer: 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8
or
PBS (20 mM sodium phosphate, 280 mM NaCl, 6 mM potassium chloride), pH 7.4
Elution buffer:
2.5 mM desthiobiotin in binding buffer
Regeneration buffer: 0.5 M NaOH
or
1 mM HABA (2-[4’-hydroxy-benzeneazo] benzoic acid) in binding buffer

See Appendix 4 (Characteristics of StrepTactin Sepharose® High Performance products) for details on regeneration of the medium.

Water and chemicals used for buffer preparation should be of high purity. Filter buffers through a 0.22 µm or a 0.45 µm filter before use.


Purification

The recommended flow velocity is 150 cm/h.

  1. Remove the stoppers and connect the column to the system. Avoid introducing air into the column.
  2. If the column has been stored in 20% ethanol, wash out the ethanol with at least 5 column volumes of distilled water or binding buffer at a flow velocity of 50 to 100 cm/h.
  3. Equilibrate the column with at least 5 column volumes of binding buffer.
  4. Apply the pretreated sample.
  5. Wash with 5 to 10 column volumes of binding buffer or until no material appears in the effluent.
  6. Elute with ~6 column volumes of elution buffer. The eluted fractions can be buffer exchanged using a prepacked desalting column (see Chapter 11, Desalting/buffer exchange and concentration).
  7. After elution, regenerate the column by following the procedure described in Appendix 4 (Characteristics of StrepTactin Sepharose® High Performance products).

Scale-up is typically performed by keeping bed height and flow velocity (cm/h) constant while increasing bed diameter and volumetric flow rate (ml/min). See Figure 7.4 (Purification using StrepTrap HP 1 ml and 5 ml) for an example of scale-up using this medium.

Store StrepTactin Sepharose® High Performance in 20% ethanol at 2°C to 8°C. After storage, equilibrate with binding buffer before use.

Materials

     
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