Characteristics of Dextrin Sepharose® High Performance Products

Appendix 3, Extracted from Affinity Chromatography Vol. 2: Tagged Proteins, GE Healthcare, 2016
 

This robust, high-resolution chromatography medium is based on the 34 µm Sepharose High Performance matrix. Due to the small size of the beads, the MBP-tagged protein is eluted in a narrow peak, minimizing the need for further concentration steps. Purification is performed under physiological conditions, and mild elution using maltose preserves the activity of the target protein. These mild conditions may even allow purification of intact protein complexes. Dextrin Sepharose High Performance tolerates all commonly used aqueous buffers and is easily regenerated using 0.5 M sodium hydroxide (see later in this appendix).
Table A3.1 summarizes key characteristics of Dextrin Sepharose High Performance. Table A3.2 summarizes the characteristics of this chromatography medium in prepacked columns.
Details on regeneration of the medium and columns follow.

Table A3.1.
Characteristics of Dextrin Sepharose High Performance

Matrix Rigid, highly cross-linked 6% agarose
Average particle size 34 µm
Ligand Dextrin
Dynamic binding capacity1 Approx. 7 mg MBP2*-paramyosin-δ-Sal/ml medium (Mr ~70 000,  multimer in solution)
Approx. 16 mg MBP2*-β-galactosidase/ml medium (Mr ~158 000,  multimer in solution)
Recommended flow velocity2 ≤ 150 cm/h
Maximum flow velocity2 < 300 cm/h
Maximum back pressure2 0.3 MPa, 3 bar
Chemical stability3 Stable in all commonly used aqueous buffers, 0.5 M NaOH  (regeneration and cleaning)
pH stability, working range
short-term
> 7
2 to 13
Storage 2°C to 8°C in 20% ethanol

1 Binding capacity is protein dependent.
2  H2O at room temperature.
3  The presence of reducing agents, e.g., 5 mM DTT, may decrease yield. Higher ionic strength does not decrease affinity because MBP binds to dextrin primarily by hydrogen binding. Agents that interfere with hydrogen binding, such as urea and Gua-HCl, are not recommended. The presence of 10% glycerol may decrease the yield, and 0.1% SDS completely eliminates the binding.
 
 
Table A3.2. Characteristics of MBPTrap HP

Chromatography medium Dextrin Sepharose High Performance
Average particle size 34 µm
Dynamic binding capacity1 Approx. 7 mg MBP2*-paramyosin δ-Sal/ml medium (Mr ~70 000,  multimer in solution)
Approx. 16 mg MBP2*-β galactosidase/ml medium (Mr ~158 000,  multimer in solution)
Column volume 1 ml or 5 ml
Column dimensions 0.7 × 2.5 cm (1 ml)
1.6 × 2.5 cm (5 ml)
Recommended flow rates 1 and 5 ml/min for 1 and 5 ml columns, respectively
Maximum flow rates 4 and 20 ml/min for 1 and 5 ml columns, respectively
Maximum back pressure2 0.3 MPa, 3 bar
Chemical stability3 Stable in all commonly used aqueous buffers
pH stability, working range
short-term
> 7
2 to 13
Storage 2°C to 8°C in 20% ethanol

1 Binding capacity is protein dependent.
2 H2O at room temperature.
3 The presence of reducing agents, e.g. 5 mM DTT, may decrease yield. Higher ionic strength does not decrease affinity because MBP binds to dextrin primarily by hydrogen binding. Agents that interfere with hydrogen binding, such as urea and Gua-HCl, are not recommended. The presence of 10% glycerol may decrease the yield, and 0.1% SDS completely eliminates the binding.

Cleaning of Dextrin Sepharose products

After purification, the medium should be regenerated as follows:

  1. Regenerate the column with 3 column volumes of distilled water followed by 3 column volumes of 0.5 M NaOH and 3 column volumes of distilled water. For bulk Dextrin Sepharose HP medium, use a flow velocity of 75 to 150 cm/h. For MBPTrap columns, use 0.5 to 1.0 ml/min for the 1 ml columns or 2.5 to 5.0 ml/min for the 5 ml columns for NaOH, and 1 ml/min or 5 ml/min, respectively, for distilled water.
  2. Re-equilibrate the column with 5 column volumes of binding buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4) before starting the next purification.

An alternative to the above regeneration is to replace 0.5 M NaOH with 0.1% SDS.  Do not regenerate with 0.1% SDS in a cold-room since the SDS may precipitate.

If P-1 pump is used, a maximum flow rate of 1 to 3 ml/min can be run on a  MBPTrap HP 1 ml column.

 

Materials

     
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