How could a serum separation experiment do without it?

　　 Centrifuge Widely used in serum separation experiments.

　　Serum proteins are primarily composed of albumin and globulins, each performing its own important functions.

　　Complete blood clotting and centrifugation conditions are two critical steps. For centrifugation, a horizontal centrifuge must be used. The specific operational procedure is as follows: Immediately after blood collection, gently invert the blood collection tube 4–5 times to thoroughly mix the sample. Allow the sample to fully clot for 30 minutes. Set the centrifuge radius to 200 mm and maintain the centrifuge speed at 3,500–4,000 rpm for 10 minutes. The serum will be completely separated from the blood clot by the separating gel. The serum sample can either be directly loaded onto the analyzer for testing or transferred into the instrument’s dedicated sample cups. Only under these conditions can high-quality serum samples be prepared, indicating that the separating gel performs effectively.

　　After protein precipitation by salting out, the resulting protein precipitate, when dissolved in water, retains the biological activity of the protein. Therefore, the salting-out method is the most commonly used technique for studying the structure and function of protein molecules. In addition to the salting-out method, other commonly used techniques for protein separation include precipitation with organic solvents, isoelectric point precipitation, column chromatography, and ultracentrifugation.

　　Experimental equipment and reagents

　　1. Equipment: 10 ml stoppered graduated test tubes, centrifuge, centrifuge tubes, microtest tubes, 5 ml pipettes, two droppers, three sets of petri dishes, cellulose acetate membranes, spotting devices, electrophoresis tank, electrophoresis apparatus, and a 1.0 × 20 cm chromatography column.

　　2. Reagents:

　　(1) Sephadex G-25 dextran gel: Weigh 10 g of Sephadex G-25 and swell it in 200 ml of 0.02 mol/L phosphate buffer at pH 6.5 for 24 hours.

　　(2) Physiological saline: Weigh 0.89 g of analytical-grade NaCl and dilute it with distilled water to a final volume of 100 ml.

　　(3) Saturated ammonium sulfate solution: At 25℃, weigh approximately 767 g of (NH₄)₂SO₄ and dissolve it in 1000 ml of distilled water until no further dissolution is possible. This constitutes the saturated ammonium sulfate solution.

　　(4) Barbiturate buffer (pH 8.6): Weigh 15.458 g of sodium barbiturate and 2.768 g of barbiturate, then dilute to 1000 ml with distilled water.

　　(5) Amino Black 10B Staining Solution: Weigh 0.5 g of Amino Black 10B, add it to 100 ml of rinsing solution, and continuously shake until completely dissolved.

　　(6) Rinsing solution: Take 450 ml of 95% ethanol and 100 ml of glacial acetic acid, mix them thoroughly, and then dilute with distilled water to a final volume of 1000 ml.

　　Salting out to precipitate proteins:

　　Take one 10-mL stoppered graduated test tube and add 2.5 mL of serum solution. Slowly add an equal volume of saturated (NH4)2SO4 solution, shake thoroughly until a precipitate forms, and let the mixture stand for 10 minutes. Filter through double-layer filter paper, discarding the precipitate and collecting the filtrate.

　　Protein molecules can remain stable in aqueous solutions thanks to two stabilizing factors: the hydration shell and surface charge. When these stabilizing factors are disrupted, protein molecules can aggregate and precipitate out of solution. There are many methods for precipitating proteins; depending on the specific mechanisms used to disrupt the stabilizing factors, these include the neutral salt precipitation method, the organic solvent precipitation method, the heavy-metal salt precipitation method, and the alkaloid reagent precipitation method. The salt precipitation method involves using a neutral salt—such as ammonium sulfate ((NH₄)₂SO₄)—to disrupt the protein's hydration shell, thereby causing the proteins to aggregate and precipitate. Different proteins require different concentrations of neutral salts for precipitation, and at varying salt concentrations, the solubility of proteins differs. Globulins tend to precipitate when the salt concentration reaches about half-saturation, whereas albumins precipitate only when the salt concentration is fully saturated. By taking advantage of this difference in precipitation behavior, proteins can be fractionally precipitated. This salt precipitation technique is known as fractional salt precipitation.

　　Precautions for this experiment:

　　1. Only personnel who have undergone rigorous training are permitted to operate the centrifuge.

　　2. Wear gloves and protective devices for the eyes and mucous membranes when operating.

　　3. Standardized laboratory techniques can help prevent or minimize the generation of splashes and aerosols. Blood and serum should be carefully pipetted rather than poured. Sucking liquids into the mouth is strictly prohibited.

　　4. After use, pipettes should be completely immersed in an appropriate disinfectant solution. The pipettes should be soaked in the disinfectant solution for an adequate period of time before being discarded or sterilized and then reused after cleaning.

　　5. Discarded specimen tubes containing blood clots or other materials should, after being capped, be placed in an appropriate leak-proof container and subjected to high-pressure sterilization and/or incineration.

　　6. Appropriate disinfectants should be available for cleaning up splashed and spilled specimens.