Performing a Purification with Calmodulin Sepharose 4B

Extracted from Affinity Chromatography Principles and Methods, GE Healthcare, 2007

Calmodulin is a highly conserved regulatory protein found in all eukaryotic cells. This protein is involved in many cellular processes such as glycogen metabolism, cytoskeletal control, neurotransmission, phosphate activity and control of NAD+/NADP+ ratios. Calmodulin Sepharose 4B provides a convenient method for the isolation of many of the calmodulin binding proteins involved in these pathways.

Calmodulin binds proteins principally through their interactions with hydrophobic sites on its surface. These sites are exposed after a conformational change induced by the action of Ca2+ on separate Ca2+-binding sites. The binding of enzymes may be enhanced if the enzyme substrate is present and enzyme-substrate-calmodulin-Ca2+ complexes are particularly stable.

Purification Options

  Binding capacity/ml medium Maximum operating flow Comments
Calmodulin Sepharose 4B No data available 75 cm/h* 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.

Performing a Separation

Binding buffer: 50 mM Tris-HCl, 0.05–0.2 M NaCl, 2 mM CaCl2, pH 7.5

Elution buffer: 50 mM Tris-HCl, 0.05–0.2 M NaCl, 2 mM EGTA, pH 7.5

  1. Pack the column (see Appendix 3, Column packing and preparation) and wash with at least 10 column volumes of binding buffer to remove preservative.
  2. Equilibrate the column with 10 column volumes of binding buffer.
  3. Apply the sample, using a low flow from 15 cm/h, during sample application (flow rate is the most significant factor for maximum binding).
  4. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A280 nm).
  5. Elute with 5 column volumes of elution buffer.

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 54, Purifcation or removal of serine proteases, e.g. thrombin and trypsin, and zymogens).

Remove free calmodulin from the sample by hydrophobic interaction chromatography in the presence of Ca2+ 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 Ca2+ 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 CaCl2. Continue washing with 3 column volumes of 0.1 M sodium acetate buffer, 2 mM CaCl2, 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  coupled to Sepharose 4B by  the CNBr method. Short term 4–9 Long term 4–9 90 µm

* 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.

Materials

     

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