Purification using Dextrin Sepharose® High Performance

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

Dextrin Sepharose® High Performance is a robust, high-resolution chromatography medium based on the 34 µm Sepharose® High Performance. The small, evenly sized beads ensure that MBP-tagged proteins elute in narrow peaks, thus minimizing the need for further concentration steps. Dextrin Sepharose® High Performance tolerates all commonly used aqueous buffers and is easily regenerated using 0.5 M NaOH, allowing the same column to be used for repeated purifications. Table A3.1 (see Appendix 3, Characteristics of Dextrin Sepharose® High Performance products) summarizes the characteristics of Dextrin Sepharose® High Performance.


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:
Optional:
Elution buffer:
Regeneration buffer:
20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4
1 mM DTT
10 mM maltose in binding buffer
0.5 M NaOH (see Appendix 3, Characteristics of Dextrin Sepharose® High Performance products) or 0.1% SDS

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. A lower flow rate can be used during sample application to optimize performance.
  5. Wash with 5 to 10 column volumes of binding buffer or until no material appears in the effluent.
  6. Elute with 5 column volumes of elution buffer. The buffer conditions of the eluted fractions can be adjusted 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 3 (Characteristics of Dextrin 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 6.7 for an example of scale-up using this medium.

Store Dextrin 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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