食品分析(英文课件)：Protein Amino acid Analysis.pptVIP

Procedures of lowry’s Method 1. Dilute protein sample to contain 20-100 μg. 2. K Na tartrate-Na2CO3 solution is added after cooling and incubated at RT for 10 min. 3. CuSO4- K Na tartrate-NaOH solution is added after cooling and incubated at RT for 10 min. 4. Freshly prepared Folin Reagent is added, then the reaction mixture is mixed and incubated at 50 deg.C for 10 min. 5. Absorbance is read at 650 nm. 6. A standard curve of BSA is carefully constructed for estimating protein conc of the unknown. Applications: Widely used in protein biochemstry, because of its simplicity and sensitivity. But not widely used in Food proteins analysis without extracting proteins from the food mixtrue. Advantages: 1. 50-100 times more sensitive than biuret method, and 10-20 times than 280 nm UV absorption method. 2. Less affected by turbidity of the sample. 3. More specific than most other method. 4. Relatively simple, can be done in 1-1.5 hours. Disadvantages: 1. Color varies with different proteins to a greater extent than the biruet method. 2. Color is not strictly proportional to protein conc. 3. The reaction is Interfered with to a varying degree by sucrose, lipids, phosphate buffers, monosaccharides, and hexoamines. 4. high conc., of reducing sugars, ammonium salfate, and sulfhydryl compounds interfere with the reaction. Properties of lowry’s Method Protein Determination with the BCA Reagent Reagent A: 1% Na bicinchoninate (BCA), 2% , Na2CO3, 0.95% NaHCO3, 0.16% Na tartrate, and 0.4% NaOH, pH 11.25. Reagent B: 4% CuSO4. To prepare working solution, mix 50 ml of reagent A and 1 ml of reagent B. Principle: This method is based on the interaction of Cu2+ ions with proteins under alkaline conditions. This leads to Cu2+ transition into Cu+ ions, and the latter are detected by BCA in a highly sensitive reaction. In this reaction, reagent green color changes into purple and the staining intensity is proportional to protein concentration in the solution (562nm). This method may