Characteristics of StrepTactin Sepharose® High Performance Products

Appendix 4, 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, Strep-tag II protein is eluted in a narrow peak, minimizing the need for further concentration steps.

Purification is performed under physiological conditions, and mild elution using desthiobiotin preserves the activity of the target protein. The mild conditions even allow purification of intact protein complexes.

Table A4.1 summarizes key characteristics of StrepTactin Sepharose® High Performance, Table A4.2 lists the compatibility of the chromatography medium with various additives, and Table A4.3 summarizes the characteristics of this medium in prepacked columns.

Details on regeneration of the medium and columns follow.
 
Table A4.1. Characteristics of StrepTactin Sepharose® High Performance

Matrix Rigid, highly cross-linked agarose
Average particle size 34 µm
Ligand StrepTactin
Ligand concentration Approx. 5 mg/ml medium
Dynamic binding capacity1 Approx. 6 mg Strep-tag II protein/ml medium
Max. flow velocity2 300 cm/h
Recommended flow velocity2 ≤ 150 cm/h
Maximum back pressure2 0.3 MPa, 3 bar
Chemical stability Stable in all commonly used buffers, 0.5 M NaOH (regeneration and cleaning), reducing agents and detergents (see Table A4.2)
pH, working range pH 7.0
Storage 2°C to 8°C in 20% ethanol

1 Dynamic binding capacity (DBC) is defined as mg protein applied per ml chromatography medium at the point where the concentration of protein in the column effluent reaches a value of 10% of the concentration in the sample. DBC was tested here with GAPDH-Strep-tag II Mr 37 400. Binding capacity is protein dependent.
2 H2O at room temperature.
 
Table A4.2. Compatibility of StrepTactin Sepharose® High Performance with different additives1

Additive2 Concentration
Reduction agents  
DTT 50 mM
β-mercaptoethanol 50 mM
Non-ionic detergents  
C8E4, Octyltetraoxyethylene
C10E5, Decylpentaoxyethylene
C10E6
C12E8
C12E9, Dodecyl nonaoxyethylene (Thesit)
Decyl-β-D-maltoside
N-dodecyl-β-D-maltoside
N-nonyl-β-D-glucopyranoside
N-octyl-β-D-glucopyranoside
Tween 20
max. 0.88%
0.12%
0.03%
0.005%
0.023%
0.35%
0.007%
0.2%
2.34%
2%
Ionic detergents  
N-lauryl-sarcosine
8-HESO;N-octyl-2-hydroxy-ethylsulfoxide
SDS
2%
1.32%
0.1%
Zwitterionic detergents  
CHAPS
DDAO
LDAO
0.1%
0.034%
0.13%
Others  
Ammonium sulfate
CaCl2
EDTA
Guanidine
Glycerol
Imidazole
MgCl2
Urea
NaCl
2 M
max. 1 M
50 mM
max. 1 M
max. 25%3
500 mM4
1 M
max. 1 M
5 M

1 Data kindly provided by IBA GmbH, Germany, the manufacturer and IP owner of the Strep-Tactin ligand.
2 The additives have been successfully tested for purifying GADPH-Strep-tag II with concentrations up to those listed. Higher concentrations may, however, be possible for reagents not marked with “max.” Since binding depends on the sterical accessibility of Strep-tag II in the context of the particular protein, the possible concentration may deviate from the given value for other proteins.
3 Yield may decrease.
4 500 mM imidazole in sample tested by GE.
 
Table A4.3. Characteristics of StrepTrap HP

Chromatography medium StrepTactin Sepharose® High Performance
Average particle size 34 µm
Ligand concentration Approx. 5 mg/ml medium
Dynamic binding capacity1 Approx. 6 mg Strep-tag II protein/ml medium
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 stability Stable in all commonly used buffers, reducing agents, and detergents  (see Table A4.2)
pH, working range > pH 7.0
Storage 2°C to 8°C in 20% ethanol

1 Binding capacity is protein to protein dependent. Dynamic binding capacity (DBC) was tested here with GADPH-Strep-tag II, Mr 37 400.
2 H2O at room temperature.
 
Regeneration and cleaning of StrepTactin Sepharose® High Performance
Recommended flow velocity is 75-150 cm/h.

  1. Regenerate and clean 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.
  2. Re-equilibrate the column with 5 column volumes of 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) before starting the next purification.

An alternative to the above regeneration/re-equilibration is 15 column volumes of 1 mM HABA (2-[4’-hydroxy-benzeneazo] benzoic acid) in binding buffer followed by 30 column volumes of binding buffer. The displacement is detected by the change in color of the medium in the column from yellow to red. This color change is due to the accumulation of HABA/StrepTactin complexes. The HABA is washed away with the binding buffer.
 
Regeneration and cleaning of StrepTrap HP 1 ml and 5 ml

  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. Use a flow rate of 0.5 to 1 ml/min or 2.5 to 5 ml/min for 1 ml and 5 ml columns, respectively, with 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 before starting the next purification.

An alternative to the above regeneration/re-equilibration is 15 column volumes of 1 mM HABA (2-[4’-hydroxy-benzeneazo] benzoic acid) in binding buffer followed by 30 column volumes of binding buffer. Use a flow rate of 2 ml/min or 10 ml/min for 1 ml and 5 ml columns, respectively. The displacement is detected by the change in color of the medium in the column from yellow to red. This color change is due to the accumulation of HABA/StrepTactin complexes. The HABA is washed away with the binding buffer.

If P-1 pump is used, a maximum flow rate of 1 to 3 ml/min can be run on a HiTrap 1 ml column packed with Sepharose® High Performance.

Materials

     
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