Affinity Chromatography - Department of Molecular and Cellular ...

Remove proteases as quickly as possible from the sample as the calmodulin-binding sites on
proteins are frequently very susceptible to protease action (see page 53).
Remove free calmodulin from the sample by hydrophobic interaction chromatography in
the presence of Ca 2+ on HiTrap Phenyl FF (high sub) or by ion exchange chromatography
on HiTrap Q FF.
Since some non-specific ionic interactions can occur, a low salt concentration
(0.05–0.20 M NaCl) is recommended to promote binding to the ligand while eliminating
any non-specific binding.
Use chelating agents to elute the proteins. Chelating agents strip Ca 2+ from the calmodulin,
reversing the conformational change that exposed the protein binding sites. Calcium ions
may also be displaced by a high salt concentration, 1 M NaCl.
Cleaning
Alternative 1
Wash with 3 column volumes of 0.05 M Tris-HCl, 1.0 M NaCl, 2 mM EGTA, pH 7.5 and
re-equilibrate immediately with 5–10 column volumes of binding buffer.
Alternative 2
Wash with 3 column volumes of 0.1 M ammonium carbonate buffer, 2 mM EGTA, pH 8.6
followed by 3 column volumes of 1 M NaCl, 2 mM CaCl 2 . Continue washing with 3 column
volumes of 0.1 M sodium acetate buffer, 2 mM CaCl 2 , pH 4.4 followed by 3 column volumes
of binding buffer.
Remove severe contamination by washing with non-ionic detergent such as 0.1%
Triton X-100 at +37 °C for 1 min.
Media characteristics
Ligand density Composition pH stability* Mean particle
size
Calmodulin Sepharose 4B 0.9–1.3 mg/ml Bovine testicular calmodulin Short term 4–9 90 µm
coupled to Sepharose 4B by Long term 4–9
the 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.
Chemical stability
Stable in all commonly used aqueous solutions.
Storage
Wash media and columns with 20% ethanol (use approximately 5 column volumes for
packed media) and store at +4 to +8 °C.
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