Phenyl-Sepharose chromatographic assay of calmodulin

Summary

This protocol has been developed over many years of practice, and it is not an example of a first attempt to purify a protein by HIC chromatography. For calmodulin, phenyl-Sepharose is the preferred HIC packing material. This experiment was derived from the Experimental Guide to Protein Purification and Characterization, by Hauchu Zhu.

Operation method

Phenyl-Sepharose chromatographic assay of calmodulin

Materials and Instruments

Calcimodulin-containing components CaCl2 (solid) Ammonium bicarbonate Buffer E Buffer F
Dialysis bags Phenyl-Sepharosundefined4B Packings Glass chromatography columns Peristaltic pumps Ultraviolet detectors, segment collectors, and borosilicate glass tubes Freeze dryers Freeze dryers

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Materials and equipment

Calmodulin-containing fractions, collected from DEAE columns and combined (see Experiment 4)

Dialysis bags (nominal MWCO 6000-8000 Da; washed)

Phenyl-Sepharosundefined4B packing (solubilized (Pharmacia Biotech, Inc.))

Glass chromatography column (2.5 cmX10 cm diameter)

_CaCl2 (solid)

Chromatography setup: peristaltic pump, UV detector, segment collector and borosilicate glass tubes (13x100 mm)

Ammonium bicarbonate (10 mmol/L;pH 8.0) (8000 ml)

Lyophilizer/freeze dryer

Reagents

Buffer E (500 ml)

Buffer E (200 ml) with 0.2mol/L NaCl

Buffer E with 8mol/L urea (100 ml)

Buffer F (500 ml)

Buffer F (200 ml) containing 0.2mol/L NaCl

(For the recipe, see "Preparation of reagents", PP.82-83)

Operating Procedures

I. Dialysis Dialysis

1) Wearing gloves, transfer the calmodulin-containing fractions consolidated from the DEAE column to the dialysis bag. Tie the ends of the dialysis bag with at least a double knot or use a clamp to tighten.

2) Dialyze against Buffer F overnight.

II. Hydrophobic Interaction Chromatography

1) Wash the Phenyl-Sepharose Packing with Buffer E (without NaCl) to remove the ethanol used to store the packing. Store in buffer E as a 50% suspension.

2) Load about 40 ml of Phenyl-Sepharose Packing onto a 2.5x10-cm glass column. Equilibrate the column by drenching several column volumes with Buffer F (without NaCl) at a low flow rate of about 2 ml/min.

3) Carefully transfer the reservoir solution from the dialysis bag to a beaker. Note the volume. Reserve about 1% for analysis and note the volume. Add solid _CaCl2 to a final concentration of 1 mmol/L to ensure that the calcium ions are saturated, regardless of what is contained in the dialyzed sample.

4) Load the phenyl-Sepharose column and allow the sample to bind for 30 to 60 minutes during the loading process.

5) Wash the column with buffer F containing 0.2 mol/L NaCl for about 2~4 volumes (60~120 ml). Collect the wash solution in a beaker or flask.

6) Remove all buffer from the top of the column. Elute with Buffer E containing 0.2mol/L NaCl and collect 2~4-ml of component by division. Monitor UV absorption and stop when the peak returns to baseline.

7) Wash the column thoroughly with Buffer E containing 8md/L urea. Regenerate with Buffer E and store the column.

8) Analyze each component by SDS-polyacrylamide gel electrophoresis for calmodulin eluates. typical results of the HIC assay are shown in Figures 1-8.

9) Combine the calmodulin-containing fractions, dialyze against 10 mmol/L ammonium bicarbonate (pH 8.0), and lyophilize. The final dried calmodulin sample was weighed. Pure calmodulin is typically obtained at 25-40 mg per kg (wet weight) of gizzard tissue. Although ammonium bicarbonate volatilizes readily, there may be some dry weights in the sample that are not protein. The sample can be dissolved in water and then lyophilized. Alternatively, pure water can be thoroughly dialyzed, but under such conditions precipitation may occur and result in a mechanical loss of protein recovery.

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