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Rapid detection method for proteins

2023-12-27 17:39

1. Principle

When urea is carefully heated to 150-160 ℃, an ammonia molecule can be removed between two molecules to form a diurea (also known as biurea). Biurea reacts with a base and a small amount of copper sulfate solution to form a purple red complex, which is called the double urea reaction. Due to the presence of plate bonds (- CO-NH -) in protein molecules, which have a similar structure to biuret, this reaction can also occur to generate purple red complexes. Under certain conditions, the color depth is directly proportional to the protein content. Based on this, the protein content can be determined by absorption spectrophotometry. The maximum absorption wavelength of this complex is 560mm.

2. Reagents

(1) Alkaline copper sulfate solution

① Using glycerol as a stabilizer: Add 10mL of 10mol/L KOH and 3mL of glycerol to 937mL of distilled water, stir vigorously, and slowly add 50mL of 40g/L copper sulfate (CuSO4 · 5H2O) solution.

② Using potassium sodium tartrate as a stabilizer: Add 10mL 10mol/L KOH and 20mL 250g/L potassium sodium tartrate solution to 930mL distilled water, stir vigorously, and slowly add 40mL 40g/L copper sulfate solution.

When preparing reagents and adding copper sulfate solution, vigorous stirring is necessary, otherwise copper hydroxide precipitation will be generated.

(2) Carbon tetrachloride (CCl4).

3. Instruments

(1) Spectrophotometer.

(2) Centrifuge (4000r/min).

4. Operation steps

(1) The standard curve is drawn using a sample with protein content measured using the Kjeldahl method as the standard protein sample. Weigh and mix standard protein samples with protein content of 40, 50, 60, 70, 80, 90, 100, and 110mg into 8 50mL Nessler tubes. Then add 1mL of carbon tetrachloride to each tube and accurately dilute 50mL with alkaline copper sulfate solution (① or ②). Shake for 10 minutes and let it stand for 1 hour. Centrifuge the upper layer of clear liquid for 5 minutes. Take the transparent solution separated by centrifugation and place it in a colorimetric dish. Adjust the instrument zero point with distilled water as the reference solution at a wavelength of 560nm and measure the absorbance A of each solution. Draw a standard curve with protein content as the x-axis and absorbance A as the y-axis.

(2) Accurately weigh an appropriate amount of the sample (such that the protein content is between 40-110mg) into a 50mL Nessler's colorimetric tube, add 1mL of carbon tetrachloride, follow the above steps for color development, and measure its absorbance A under the same conditions. The protein quality can be obtained by using the measured A value on the standard curve, and then the protein content can be obtained.

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