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Biuret method Reagents Dissolve 4.Og sodium hydroxide in about 400ml of distilled water. Add 4.5g sodium potassium tartrate. Mix to dissolve. Then add 1.5g CuSO4.5H20 followed by 4.5g potassium iodide. Transfer the solution into a 500ml volumetric flask and make up to the mark with distilled water. Store in a tightly stoppered polyethylene bottle at room temperature (25-35°C). Stable for 6 months. Stock bovine immunoglobulin (Blg) solution (10mg/ml). Dissolve 0.2g Blg in 20ml distilled water. Working Big concentration range: 0.5-1.25 mg/ml Method 1. Prepare the following test tubes and perform the method as indicated: Table 2: Preparation of test tubes for the biuret method Blank Tube Tube Tube Tube Tube Unknown 1 2 3 4 5 Distilled 1.0 ml 0.5ml H20 0.25 1.0 mg/ml ml Blg 0.5 mg/ml 1.0 Big ml 0.75 1.0 mg/ml ml Blg
Unknown Tube 4 Tube 5 0.5ml 이 Table 2: Preparation of test tubes for the biuret method Blank Tube Tube Tube 1 2 3 Distilled 1.0 ml H20 0.25 1.0 mg/ml ml Big 0.5 mg/ml 1.0 Blg ml 0.75 1.0 mg/ml ml Big 1.0 mg/ml Blg 1.25 mg/ml Blg Unknown protein Biuret 4.0ml 4.Oml 4.Oml 4.Oml reagent 2. Mix thoroughly after each addition. 3. Incubate at room temperature for 30 minutes. 4. Read absorbance at 540nm for all the tubes. 1.0 ml 1.0 ml 0.5ml 4.Oml 4.Oml 4.Oml
Calculate the protein concentration of an unknown sample (in mg/ml) with an absorbancy value of 0.427 using the standard curve below. 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 Absorbancy 540nm 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.0 0.1 02 0.3 0.4 0.5 1.1 1.2 13 1.4 1.5 1.6 0.6 0.7 0.8 0.9 10 Big concentration (mg/ml)