Affinity Chromatography - Department of Molecular and Cellular ...

Elution buffers:
• use low concentrations of the free cofactor, NAD + or NADP + (up to 20 mM), or increase ionic strength
up to 2 M NaCl or 1 M KCl.
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 with 2 column volumes of 6 M guanidine
hydrochloride or 8 M urea. Alternatively, wash the column with 2 column volumes of
detergent in a basic or acidic solution, e.g. 0.1% Triton X-100 in 1 M acetic acid. Remove
residual detergent by washing with 5 column volumes of 70% ethanol. In both cases wash
immediately with 5 column volumes of binding buffer.
Media characteristics
Ligand and density Composition pH stability* Mean particle
size
Red Sepharose CL-6B Procion Red HE 3B Ligand coupled to Short term 3–13 90 µm
2 µmoles/ml Sepharose CL-6B using Long term 4–12
the triazine 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.
Chemical stability
Stable with all commonly used aqueous buffers and additives such as 8 M urea and
6 M guanidine hydrochloride.
Storage
Store freeze-dried powders under dry conditions and below +8 °C.
Wash media and columns with 20% ethanol in 0.1 M KH 2 PO 4 , pH 8.0 (use approximately
5 column volumes for packed media) and store at +4 to +8 °C.
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