Affinity Chromatography - Department of Molecular and Cellular ...

Purification options
Binding capacity/ml Maximum Comments
medium
operating flow
2'5' ADP Sepharose 4B Glucose-6-phosphate, 75 cm/h* High specificity for proteins
dehydrogenase, 0.4 mg with affinity for NADP + .
(0.1 M Tris-HCl, 5 mM EDTA, Supplied as a freeze-dried
1 mM 2-mercaptoethanol powder, rehydration required.
buffer, pH 7.6).
Red Sepharose CL-6B Rabbit lactate dehydrogenase, 150 cm/h* General specificity for proteins
2 mg with affinity for NADP + and
other proteins that react less
specifically.
Supplied as dry powder,
rehydration required.
*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.
2'5' ADP Sepharose 4B
Purification example
Figure 45 shows a linear gradient elution used for the initial separation of NADP + -dependent
enzymes from a crude extract of Candida utilis.
B
Enzyme activity
A
C
D
E
0.8
0.6
0.4
0.2
NADP concentration (mM)
+
0 20 40 60 80 100 120
Fraction number
Fig. 45. Gradient elution with 0–0.6 mM NADP + . A: non-interacting protein, B: glucose-6-phosphate dehydrogenase,
C: glutamate dehydrogenase, D: glutathione reductase, E: 6-phosphogluconate dehydrogenase.
(Brodelius et al., Eur. J. Biochem. 47, 81–89 (1974)).
Performing a separation
Swell the required amount of powder for 15 min. in 0.1 M phosphate buffer, pH 7.3 (100 ml
per gram dry powder) and wash on a sintered glass filter (porosity G3). 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.
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