Affinity Chromatography - Department of Molecular and Cellular ...

Media characteristics
Ligand density Composition pH stability* Mean particle
size
5' AMP Sepharose 4B 2 µmoles/ml N 6 (6-aminohexyl-) 5' AMP Short term 4–10 90 µm
coupled to Sepharose 4B Long term 4–10
using 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.
**The attachment of the ligand via an alkyl linkage to the N 6 amino group gives a stable product that is conformationally
acceptable to most 5' AMP- or adenine nucleotide cofactor-requiring 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.
HiTrap Blue HP, Blue Sepharose 6 Fast Flow
The information supplied for the purification or removal of albumin (page 69) is applicable
also to the purification of enzymes with an affinity for NAD + .
Performing a separation
As for albumin (see page 71), but note the following:
For elution use low concentrations of the free cofactor, NAD + or NADP + (1–20 mM), or
increase ionic strength (up to 2 M NaCl or KCl, 1 M is usually sufficient).
For less specifically bound proteins: use higher concentrations of cofactor or salt or more
severe eluents such as urea or potassium isothiocyanate. Polarity reducing agents such as
dioxane (up to 10%) or ethylene glycol (up to 50%) may be used.
NADP+-dependent dehydrogenases and other enzymes with
affinity for NADP+
2'5' ADP Sepharose 4B, Red Sepharose CL-6B
NADP + -dependent dehydrogenases interact strongly with 2'5' ADP. Selective elution with
gradients of NAD + or NADP + has allowed the resolution of complex mixtures of dehydrogenase
isoenzymes using 2'5' ADP Sepharose 4B.
Synthesis of the medium takes place in several steps. Diaminohexane is linked to 2'5' ADP
via the N 6 of the purine ring. The derivatized ADP is then coupled to Sepharose 4B via the
aminohexane spacer. Figure 43 shows the partial structure of 2'5' ADP Sepharose 4B.
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