For the purification of calmodulin, two different operations were performed for the crude graded separation of smooth muscle tissue extracts: ammonium sulfate precipitation and isoelectric point precipitation. These two operations take advantage of two different properties of calmodulin, namely its high solubility at pH above neutral and its acidic isoelectric point. This experiment is from the Laboratory Guide for Protein Purification and Identification by Houzhu Zhu.
Operation method
Crude graded separation experiments of calmodulin
Materials and Instruments
Supernatant Solid Ammonium Sulfate Concentrated Sulfuric Acid Tris Alkali Buffer B Move Materials and equipment For more product details, please visit Aladdin Scientific website.
Polypropylene beakers Magnetic stirrers and stirrers Polycarbonate centrifuge tubes Ultracentrifuge Scaled polypropylene measuring cylinders Coarse cotton cloth Polypropylene funnels Rubber starch brooms Scraper spoons Dialysis bags
supernatant from experiment 2 (about 700~1000 ml)
Solid ammonium sulfate
Polypropylene beaker (2x4000-ml)
Magnetic stirrer with stirrer
Polycarbonate centrifuge tubes (6x500-ml)
Ultracentrifuge with 3000-ml capacity rotor head
Polypropylene graduated cylinders (2x4000-ml)
Coarse cotton cloth
Polypropylene funnel (large, wide mouth type)
Concentrated sulfuric acid (diluted to 50%v/v aqueous solution)
Rubber starch broom/scraper
Tris base (1 mol/L; about 10 ml)
Polypropylene funnel (small, long stemmed)
Dialysis bag (nominal MWCO 6000-8000 Da; wash)
Reagents
Buffer B (10x) (Prepare 4000 ml of 1x Buffer B before use and add 2-mercaptoethanol to 1 mmol/L)
(For recipe, see "Preparation of Reagents", PP.82-83)
Operating Procedures
1) Using the volume of supernatant obtained in Experiment 2 (minus the aliquot taken for activity measurement), check the amount of solid ammonium sulfate required for 0% to 60% saturation on the Ammonium Sulfate Chart (see Appendix 4). This should be 361 g/L. Weigh the ammonium sulfate and pulverize the mass into a fine powder.
2) Place the supernatant in a 4000-ml polypropylene beaker with a large magnetic stirrer. Stir slowly on a large magnetic stirrer in a cold room. Add ammonium sulfate slowly and shakily within 20 min. If the solid ammonium sulfate settles to the bottom of the beaker, pause for a few minutes to allow it to dissolve. Then continue stirring for another 40 min (i.e., 60 min total).
3) Transfer the mixture to a 500-ml polycarbonate centrifuge tube, equilibrate, and centrifuge for 60 min at 4°C, 8000 r/min in an ultracentrifuge.
4) Remove the supernatant, pour into a polypropylene funnel lined with two layers of coarse cotton cloth and collect in a graduated polypropylene measuring cylinder. Discard the precipitate. Record the volume of the supernatant. Reserve 1% of the volume for later activation. You will need both volumes for calculations.
5) Transfer the supernatant to a 4000-ml polypropylene beaker. Measure with a pH meter, stirring at medium speed, and add 50% (v/v) sulfuric acid solution until pH reaches about 4.05. Try to keep the pH below 4.1 and above 4.0.
6) Place the beaker in a cold room and stir for 1h.
7) Transfer the supernatant to a 500-ml polycarbonate centrifuge tube. Centrifuge for 60 min at 4°C, 8000 r/min in an ultracentrifuge.
8) Remove the supernatant and discard. Using a rubber precipitation broom, resuspend the precipitate in a few milliliters of distilled water containing a few drops of 1 mol/L Tris base. The precipitate should be resuspended in the smallest volume.
9) Wearing gloves, transfer the suspension to the dialysis bag. Fill the dialysis bag with a small polypropylene funnel with a long stem. Exhaust excess air, leaving 50% empty (the bag should be flat and air free). Tie the bag with at least a double knot at each end or use a clamp.
10) Dialyze distilled water for 4 h at 40°C with one fluid change. Then, transfer to 4,000 ml lx of Buffer B and continue dialysis overnight (4°C). In all cases of dialysis, the dialysate was mixed by slow stirring in the beaker with a small stirrer. The stirrer should not be allowed to touch the dialysis bag or the bag will rupture.