Experiment 1: Determination of Protein Concentration Using Coomassie Brilliant Blue Staining Method
1. Objectives and Requirements 1.1 Learn and master the principles and operational methods of the Coomassie Brilliant Blue staining method for protein quantification. 1.2 Further familiarize with the usage of the spectrophotometer
2. Fundamental Principles The Coomassie Brilliant Blue staining method, established by Bradford in 1976 (also known as the Bradford method), is based on the quantitative binding of proteins with Coomassie Brilliant Blue G-250. When Coomassie Brilliant Blue G-250 binds to proteins, its maximum absorption peak shifts from 465 nm to 595 nm
2. Fundamental Principles Under conditions where Coomassie Brilliant Blue G-250 is present in excess and at a constant concentration, varying protein concentrations in the solution result in different amounts of dye molecules transitioning from the 465 nm absorption form to the 595 nm form. This transition follows a quantitative relationship. Generally, as protein concentration increases, the absorbance of the reagent at 595 nm increases nearly linearly, allowing the Coomassie Brilliant Blue G-250 staining method to be used for protein quantitation
Compared to the Lowry method, the advantages of the Coomassie Brilliant Blue method include: Simplicity: Requires only a single reagent for color development. Rapid reaction: Completes in a single step within 5 min. Reduced interference: Unaffected by many compounds (e.g., sugars, buffers, reducing agents, and chelators) that interfere with the Lowry method. Limitations exist, such as imperfect linearity. 2. Fundamental Principles
3. Experimental Procedures 3.1 Preparation of Standard Curve Prepare 6 clean, dry tubes and add reagents according to the following table: Tube No. Volume of H₂O (mL) Volume of Standard Protein Solution (mL) Total Protein Content in Tube (μg) Coomassie Brilliant Blue Reagent (mL) Abs₅₉₅ 1 1.0 0 0 5 0.000 2 0.8 0.2 50 5 ? 3 0.6 0.4 100 5 ? 4 0.4 0.6 1500 5 ? 5 0.2 0.8 200 5 ? 6 0.0 1.0 2500 5 ?
3. Procedures After adding all reagents, mix thoroughly. The absorbance at 595 nm (Abs₅₉₅) can be measured immediately using a spectrophotometer. Plot the standard curve with protein concentration as the x-axis and Abs₅₉₅ as the y-axis
3.2 Sample Measurement While preparing the standard curve, take another test tube, add 1 mL of the sample to be tested, then add 5 mL of Coomassie Brilliant Blue reagent. Mix thoroughly and measure Abs₅₉₅. Use the Abs₅₉₅ value to determine the protein concentration of the sample from the standard curve. To improve accuracy, perform a replicate measurement of the sample simultaneously. 3. Experimental Procedures
Experiment 2: the Identification of Reducing and Total Sugars in Sweet Potato Flour
1. Objectives 1.1 To identify reducing sugars and total sugars in sweet potato flour. 1.2 To understand the fundamental principles of reducing and total sugar determination. 1.3 To learn the operation methods of spectrophotometric (colorimetry) determination of reducing sugars. 1.4 To master the use of the spectrophotometer