Experimental principle
There are many methods for the determination of protein. Currently, Kjeldahl method is commonly used; colorimetric methods mainly include biuret method, Folin-phenol reagent method and dye combination method; ultraviolet spectrophotometry, etc.
Biuret method: in an alkaline solution, biuret (H2N-CO-NH-CO-NH2) reacts with divalent copper ions to form a purple-red complex. This reaction is called biuret reaction. . Any compound containing two or more amide groups (—CO—NH 2 ), or a similar group [such as —CH 2 —NH 2 , —CS—NH 2 , —C(NH)NH 2 , in the molecule, regardless of The above reactions can occur if such groups are directly linked or indirectly via a carbon or nitrogen atom. The protein molecule contains a large number of peptide bonds (—CO—NH—), and the biuret reaction can occur, and the color intensity is proportional to the amount of peptide bonds in a certain concentration range, that is, the protein content, and the protein content can be determined by colorimetry. The total serum protein content can be determined by calculation or by examining the standard curve compared to the same processed protein standard solution.
1.6mol/L sodium hydroxide solution
Weigh 240 g of sodium hydroxide (NaOH, pure grade), make 1 L with fresh distilled water, and store at room temperature in a sealed polyethylene bottle.
2. Biuret reagent
Weigh 3.0 g of copper sulfate (CuSQ·5H20) without loss of crystal water, dissolve it in 500 ml of fresh distilled water, add 9.0 g of sodium potassium tartrate (NaKC 4 H 4 O 6 .4H 2 O), and 5.0 g of potassium iodide (KI). After safely dissolving, 100 ml of a 6 mol/L sodium hydroxide solution was added, and finally distilled water was added to 1 L. Store in a sealed polyethylene bottle at room temperature for approximately 6 months.
3. Protein standard Â
Commercial serum protein standard or fixed-value control serum can be used as a standard. Mixed fresh serum can also be collected and calibrated by Kjeldahl method. After adding sodium azide preservation (the final concentration of sodium azide is 0.5~1.0g/L), it can be stored frozen.
Constant temperature water bath, 722 (721) spectrophotometer, straw, test tube, coordinate paper
1. Prepare 20g/L protein standard solution. If the concentration of protein standard solution is 70g/L, take this solution 2m1, add 5ml of fresh distilled water, and mix and serve.
2. Follow the table
Mix, place in a 37 ° C water bath for 10 min (or 25 ° C for 30 min), use 540 nm wavelength colorimetric, zero with a blank tube, read the absorbance of each tube.
3. Standard curve drawing
1-5 is a standard curve tube. After the absorbance is measured, the absorbance is plotted on the ordinate and the protein concentration is plotted on the abscissa.
4. Experimental Results Find the corresponding total protein concentration from the standard curve based on the absorbance of the assay tube.
1. In the biuret reagent, sodium potassium tartrate is added, and Cu2 forms a stable complex copper ion to prevent CuSO4·5H20 from destabilizing to form Cu(OH)2 precipitate. The ratio of sodium potassium tartrate to CuSO4·5H20 is not less than 3:1, and KI is added as an antioxidant.
2. Biuret reagent should be stored in a closed state. Prevent absorption of carbon dioxide from the air.
3. The various colors of the protein in this method are basically the same, the repeatability is good, and the temperature is almost unaffected by temperature. The only disadvantage is that the sensitivity is low.
4. Astragalus serum. Severe hemolysis has significant interference with this law.
5. The standard curve drawn by this method is a straight line passing through the origin, showing a good linear relationship within the concentration of 100g/L.Hose Reel Cart
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