Affinity Chromatography - Department of Molecular and Cellular ...

Reactivation
Pass one to two column volumes of a saturated solution (approximately 1.5 mM) of
2,2'-dipyridyl disulphide, pH 8.0 through the medium.
Prepare 2,2'-dipyridyl disulphide:
1. Make a stock solution by adding 40 mg 2,2'-dipyridyl disulphide to 50 ml buffer at room temperature and
stirring the suspension for several hours.
2. Filter off insoluble material.
3. Adjust the pH. The solution will be approximately 1.5 mM with respect to 2,2'-dipyridyl disulphide.
Cleaning
Wash with non-ionic detergent such as 0.1% Triton X-100 at +37 °C for 1 minute.
Re-equilibrate immediately with a minimum of 5 column volumes of binding buffer.
Media characteristics
Density of Composition pH stability* Mean particle
thiol groups
size
Thiopropyl 25 µmoles/ml Mixed disulphide containing Short term 2–8 90 µm
Sepharose 6B 2-thiopyridyl protecting groups Long term 2–8
attached to Sepharose 6B
through a chemically stable
ether linkage.
Activated Thiol 1 µmole/ml Mixed disulphide formed Short term 2–8 90 µm
Sepharose 4B between 2,2'-dipyridyl Long term 2–8
disulphide and glutathione
coupled to CNBr-activated
Sepharose 4B.
*Long term refers to the pH interval over which the matrix 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. When a molecule has been coupled to the thiolated matrix, the long term and short term
pH stability of the medium will be dependent upon the nature of that molecule.
Chemical stability
Stable to all commonly used aqueous buffers and additives such as detergents. Avoid azides.
Storage
Store freeze-dried powders below +8 °C.
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. Storage under nitrogen is recommended
to prevent oxidation of thiol groups by atmospheric oxygen.
Avoid using sodium azide, merthiolate or phenyl mercuric salts as bacteriostatic agents.
Azide ions will react with the 2,2'-dipyridyl disulphide groups, although low concentrations
(0.04%) have been used.
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