Affinity Chromatography - Department of Molecular and Cellular ...

If detergent or denaturing agents have been used during purification, these can also be used
in the low and high pH wash buffers.
Cleaning
Wash 3 times with 2–3 column volumes of buffers, alternating between 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). Re-equilibrate immediately with 3–5 column volumes of binding buffer.
Remove denatured proteins or lipids by washing the column with 2 column volumes of
detergent e.g. 0.1% Triton X-100 for 1 minute. Re-equilibrate immediately with 5 column
volumes of binding buffer.
Media characteristics
Ligand density Composition pH stability* Mean particle
size
2'5' ADP Sepharose 4B 2 µmoles/ml N 6 -(6-aminohexyl)adenosine Short term 4–10 90 µm
2'5' bisphosphate coupled to Long term 4–10
Sepharose 4B by CNBr method**
*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.
**Coupling via the N 6 position of the NADP + -analogue, adenosine 2'5' bisphosphate, gives a ligand that is
stereochemically acceptable to most NADP + -dependent enzymes.
Chemical stability
Stable to all commonly used aqueous buffers and additives such as detergents. Avoid high
concentrations of EDTA, urea, guanidine hydrochloride, chaotropic salts and strong
oxidizing agents. Exposure to pH > 10 may cause loss of phosphate groups.
Storage
Store freeze-dried product below +8 °C under dry conditions.
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.
Red Sepharose CL-6B
Performing a separation
Swell the required amount of powder for 15 min. and wash with distilled water on a sintered
glass filter (porosity G3). Use 200 ml water for each gram of dry powder, adding in several
aliquots. One gram of freeze-dried material gives a final volume of approximately 4 ml.
Pack a column (see Appendix 3).
Binding buffer: Use a buffer at around neutral pH since proteins bind specifically to Red Sepharose CL-6B
at this pH.
The binding capacity will depend upon parameters such as sample concentration, flow rate,
pH, buffer composition and temperature. To obtain optimal purification with respect to
capacity, determine the binding capacity over a range of different pH and flow rates.
77