DEAE SEPHADEX A-50 anion-exchange chromatography assay for calmodulin
DEAE SEPHADEX A-50 anion-exchange chromatography assay for calmodulin
The next step in the purification of calmodulin is anion exchange chromatography with DEAE resin. This step takes advantage of the fact that calmodulin is a strongly acidic protein and thus carries a large net negative charge at neutral or slightly basic pH conditions. This experiment was derived from the Protein Purification and Characterization Lab Guide by Houzhu Zhu.
Operation method
DEAE SEPHADEX A-50 anion-exchange chromatography assay for calmodulin
Materials and Instruments
DEAE Sephadeundefined A-50 (dry powder) Concentrated NaOH Dialysis Reservoir Buffer B Move Materials and equipment For more product details, please visit Aladdin Scientific website.
Ultracentrifuge Glass chromatography column Peristaltic pump, ultraviolet (UV) detector, segment collector and silica tubing
DEAE Sephadeundefined A-50 (dry powder) (Pharmacia Biotech, Inc.)
Concentrated NaOH (10mol/L)
Dialysis Residue, from Experiment 3
Ultracentrifuge
Glass chromatography column (5 cm diameter)
Chromatography unit: peristaltic pump, ultraviolet (UV) detector, segment collector and silica glass tubing (16X150 mm)
reagents
Buffer B (10x (4000 mlx of Buffer B before use, add 2-hydroxyethanol to 1 mmol/L) (for recipe, see "Preparation of Reagents", PP.82-83)
Operating Procedures
1) Dissolve DEAE Sephadex A-50 resin in 1x Buffer B overnight at 4°C or in a boiling water bath for 1 hour (h). The resin swells to approximately 30 ml/g at pH 8.0 (see Ion Exchange Chromatography, literature), so that approximately 15 g of dry resin is required to load one 400-ml column.
2) Adjust the pH of the resin suspension to 8.0 with concentrated NaOH The resin is supplied in an acidic form, so a few milliliters of NaOH are required Do not stir with a stirrer or the particles will be broken. Stir manually with a pipette or glass rod. After adjusting the pH, allow the resin to settle and supernatant to be removed. Wash the pellet three times with 1X Buffer B. Recalibrate the pH after each wash until it reaches 8.0 and remains there.
Note: DEAE-cellulose is supplied as a free base, therefore, its pH must be adjusted with concentrated acid (e.g., 12 ml/LHCl).
3) Carefully transfer the dialysis reservoir from the dialysis bag to the beaker. Transfer to a 30-ml plastic centrifuge tube. Centrifuge at 4°C for 1 h at 20,000 r/min in a high-speed centrifuge.
4) Pour the supernatant into a beaker and determine the conductivity of the sample at 4°C, comparing it to the conductivity of 1X Buffer B at 4°C. The sample must be equal to or above the conductivity of the buffer. The conductivity of the sample must be equal to or lower than the conductivity of the buffer. If it is too high, add distilled water until it is the same as Buffer B. Keep the volume as small as possible (this is why it is so important). Keep the volume as small as possible (this is why you record the volume by dialysis and not dilution. Leave 1% for analysis and record the volume.
5) Load the column and install the chromatography unit as shown in Figure 1-5. Replace the nylon mesh at the bottom if necessary. Leave about 2 cm of buffer above the column bed to facilitate gradient mixing. Allow the eluate to flow through the UV detector, making sure the baseline appears on the recorder. Adjust the partition collector to collect approximately 10 to 12-ml fractions.
6) Place the inlet port of the peristaltic pump into the beaker containing the sample, add the sample, and watch the inlet port closely so that no air is drawn in. Wash the column with 1X Buffer B until the UV signal on the recorder is close to the original baseline. Collect the effluent in a beaker. It is not necessary to collect the components before the start of the gradient.
7) The total volume of the gradient eluent is 1000 ml:500 ml of 1x Buffer B to 500 ml of Buffer B with 0.7mol/L NaCl. Note that lx Buffer B already contains 0.2 ml/L of salt, and that 14.62 g NaCl can be added to 500 ml of 1x Buffer B to prepare 0.7mol/L NaCl. Use a concentric plastic gradient mixer: high salt in the outer chamber, low salt in the inner chamber. The stirrer is placed in the inner chamber and stirred moderately.
8) Collect all gradient eluents in 10-12-ml portions in borosilicate glass tubes. Typical results of calmodulin purification by DEAE anion exchange chromatography are shown in Figures 1-6. 
