Crude graded separation experiments of calmodulin

Summary

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
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

Move

Materials and equipment

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.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/