Affinity Chromatography - Department of Molecular and Cellular ...

Purification options
Binding capacity Maximum Comments
operating flow
5' AMP Sepharose 4B Lactate dehydrogenase, 75 cm/h* High specificity for
10 mg/ml medium proteins with affinity
(0.1 M phosphate buffer, for NAD + . Supplied as
pH 7.0 at +20 °C)
a freeze-dried powder,
rehydration required.
HiTrap Blue HP Human serum albumin, 4 ml/min (1 ml column) General specificity for
20 mg/column proteins with affinity for
Human serum albumin, 20 ml/min (5 ml column) NADP + and other proteins
100 mg/column that react less specifically.
Prepacked columns.
Blue Sepharose 6 Human serum albumin, 400 cm/h* General specificity for
Fast Flow > 18 mg/ml medium proteins with affinity for
NADP + and other proteins
that react less specifically.
Supplied as a suspension
ready for column packing.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from
measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
5' AMP Sepharose 4B
Performing a separation
Swell the required amount of powder for 15 min. in 0.1 M phosphate buffer, pH 7.0
(100 ml per gram dry powder) and wash on a sintered glass filter. Pack the column
(see Appendix 3).
Binding buffer: 10 mM phosphate, 0.15 M NaCl, pH 7.3
If the protein of interest binds to the medium via ionic forces, it may be necessary to reduce
the concentration of NaCl in the binding buffer.
Elution buffers:
• use low concentrations of the free cofactor, NAD + or NADP + (up to 20 mM) with step or gradient elution.
If detergent or denaturing agents have been used during purification, these can also be used
in the high and low pH wash buffers.
Cleaning
Wash 3 times with 2–3 column volumes of buffers, alternating between high pH
(0.5 M NaCl, 0.1 M Tris-HCl, pH 8.5) and low pH (0.5 M NaCl, 0.1 M sodium acetate,
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.
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