Separation and Purification of Serum Albumin
Purpose
The purpose of this experiment is to understand the principle of ion exchange chromatography by separating and purifying serum albumin. Through this process, students will gain hands-on experience in protein purification techniques and learn how to manipulate conditions such as pH and ionic strength to achieve selective separation.Experimental Principle
Ion exchange chromatography is a widely used technique for separating proteins based on their charge properties. In this method, cation exchangers, such as carboxymethyl cellulose, and anion exchangers, such as diethylaminoethyl (DEAE) cellulose, are commonly employed. Proteins bind to the ion exchanger depending on their net charge, which is influenced by the pH of the solution and the degree of dissociation of both the protein and the ion exchanger. The binding force between the protein and the resin is determined by electrostatic interactions. The presence of salt can reduce these interactions, allowing for the elution of bound proteins. By adjusting the pH or ionic strength, different proteins can be separated based on their affinity for the ion exchanger. In this experiment, serum albumin is purified using a combination of salting out, desalting via gel filtration, and final purification by ion exchange chromatography. First, serum globulins are precipitated using ammonium sulfate, leaving albumin in the supernatant. Then, the sample is desalted using Sephadex G-25, which removes small molecules like salts while retaining larger proteins. Finally, DEAE-cellulose column chromatography is used to separate albumin from other proteins based on their charge at a specific pH. At a pH of 6.5, DEAE-cellulose carries a positive charge, and it binds negatively charged albumin. Other proteins, such as gamma-globulins, remain unbound and flow through. Increasing the salt concentration allows for the stepwise elution of different proteins, with albumin being collected at the end.Reagents and Equipment
1. Reagents: - 0.3 mol/L NH4Ac buffer (pH 6.5): Dissolve 23.13 g of ammonium acetate in 800 mL distilled water, adjust pH to 6.5 with dilute acetic acid or ammonia, and dilute to 1 L. - 0.06 mol/L NH4Ac buffer (pH 6.5): Dilute the 0.3 mol/L buffer 5 times. - 0.02 mol/L NH4Ac buffer (pH 6.5): Dilute the 0.06 mol/L buffer 3 times. - 1.5 mol/L NaCl–0.3 mol/L NH4Ac: Dissolve 87.7 g NaCl in 0.3 mol/L NH4Ac (pH 6.5) to make 1 L. - Saturated ammonium sulfate solution: Dissolve 850 g (NH4)2SO4 in 1 L distilled water, heat to 70–80°C, and let stand overnight. The supernatant is used. - 200 g/L sulfosalicylic acid. - 10 g/L BaCl2. 2. Materials: - Serum (from human or animal sources) 3. Equipment: - Chromatography columns (1 cm × 15 cm and 1 cm × 25 cm), column holder, reaction plate, pipette, water bath.Method of Operation
1. **Column Preparation** - **Sephadex G-25 Column**: Prepare the gel by swelling it in distilled water, then equilibrate with 0.02 mol/L NH4Ac buffer (pH 6.5). Pack the column carefully to ensure even distribution and no air bubbles. - **DEAE-Cellulose Column**: Treat the resin with NaOH and HCl, then equilibrate with 0.02 mol/L NH4Ac buffer. Ensure the pH is maintained at 6.5 throughout the process. 2. **Separation and Purification** - **Salting Out**: Add saturated ammonium sulfate to serum, centrifuge, and collect the supernatant containing albumin. - **Desalting**: Load the sample onto the Sephadex G-25 column, wash with buffer, and collect fractions. Use sulfosalicylic acid and BaCl2 to detect protein and salt. - **Purification**: Apply the desalted sample to the DEAE-column, elute with increasing concentrations of NH4Ac, and collect albumin fractions.Precautions
- Accurately prepare all buffers and maintain the pH at 6.5. - Avoid disturbing the column bed surface during loading or washing. - Monitor the elution carefully to avoid losing valuable fractions. - Prevent air bubbles from entering the column, as they can disrupt the flow and affect results. This experiment provides a comprehensive understanding of protein purification techniques and emphasizes the importance of precise control over experimental conditions.iPad Wireless Keyboard Case,LED Display Magic Keyboard Case,Detachable Magnetic Keyboard Case,Magnetic Keyboard Case,Rotatable Wireless Keyboard
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