Affinity Chromatography - Department of Molecular and Cellular ...

Cleaning
These procedures are applicable to Glutathione Sepharose 4 Fast Flow and Glutathione
Sepharose 4B.
1. Wash with 2–3 column volumes of alternating high pH (0.1 M Tris-HCl, 0.5 M NaCl,
pH 8.5) and low pH (0.1 M sodium acetate, 0.5 M NaCl, pH 4.5) buffers.
2. Repeat the cycle 3 times.
3. Re-equilibrate immediately with 3–5 column volumes of binding buffer.
If the medium is losing binding capacity, this may be due to an accumulation of precipitated,
denatured or non-specifically bound proteins.
To remove precipitated or denatured substances:
1. Wash with 2 column volumes of 6 M guanidine hydrochloride.
2. Wash immediately with 5 column volumes of binding buffer.
To remove hydrophobically bound substances:
1. Wash with 3–4 column volumes of 70% ethanol (or 2 column volumes of a non-ionic
detergent (Triton X-100 1%)).
2. Wash immediately with 5 column volumes of binding buffer.
Media characteristics
Spacer arm Ligand and density pH stability* Mean particle size
Glutathione Sepharose 4 10 carbon linker Glutathione Short term 6–9 90 µm
Fast Flow (GSTrap) 120–320 µmoles/ml Long term 6–9
Glutathione Sepharose 4B 10 carbon linker Glutathione Short term 4–13 90 µm
7–15 µmoles/ml Long term 4–13
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects
on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place
and sanitization procedures.
Chemical stability
No significant loss of binding capacity when exposed to 0.1 M citrate (pH 4.0), 0.1 M NaOH,
70% ethanol or 6 M guanidine hydrochloride for 2 hours at room temperature. No significant
loss of binding capacity after exposure to 1% SDS for 14 days.
Storage
Wash media and columns with 20% ethanol at neutral pH (use approximately 5 column
volumes for packed media) and store at +4 to +8 °C.
45