双缩脲法测蛋白含量Seperation of serum proteins by biuret method.ppt

3) Quantitative analysis of Proteins : The separation of serum proteins by?biuret?method Next Biochemistry Practical Materials required Spectrophotometer / photoelectric Colorimeter Centrifuge machine Water bath Standard Protein solution (0.2 %) Serum sample Pipette, Cuvette, Test tube, Measuring cylinder, weighing machine. Reagents Biuret reagent:- Weigh 1.50 gm. of cupric sulfate (CuSO1.5HzO) and 6.0 gm. of sodium potassium tartrate (NaKC4H40s.4Hz0) ; transfer to a, 1-liter volumetric flask, and dissolve in about 500 ml. of water. Add with constant swirling 300 ml. of 10 per cent sodium hydroxide (prepared from stock, carbonate-free, 65 to 75 per cent sodium hydroxide solution). Make to volume with water, mix, and store in a paraffin-lined bottle. This reagent should keep indefinitely but must be discarded if, as a result of contamination or faulty preparation, it shows signs of depositing any black or reddish precipitate. Globulin precipitants:- Sodium sulfate, 22.6 per cent; Diethyl ether. Sodium chloride solution (0.9 per cent)/ Normal saline. Procedure to Prepare a standard curve of absorbance versus protein in micrograms (or vice versa), and determine amounts from the curve. Pipette out a series of tubes 0.1, 0.2…1ml of protein solution and mark the tubes as 1, 2, ………..10 ↓ Make up to 2ml with water ↓ 3ml of biuret reagent added to each tube ↓ Mix well ↓ Heat the tubes at 37oc for 10 min ↓ Purple color develops ↓ Absorba