Principle of Biuret Protein Assay
Biuret protein assay is based on the principle that copper ions (Cu2+) react with peptide bonds in proteins, forming a complex that absorbs light at a specific wavelength. The biuret reagent used in the assay contains copper ions that react with the peptide bonds in the protein to form a purple-colored complex. The intensity of the purple color is directly proportional to the protein concentration in the sample. Thus, the amount of protein can be quantified by measuring the absorbance of the sample at a specific wavelength.
Why Perform Biuret Protein Assay?
Biuret protein assay is a simple, sensitive, and reproducible method for quantifying protein concentration. It is widely used in biochemical and biological research to determine the protein concentration in a sample. The assay can be performed on a wide range of protein samples, including purified proteins, cell lysates, and body fluids. The results obtained from the biuret protein assay can be used to perform various biochemical and biological studies, including enzyme kinetics, protein-protein interactions, and protein expression studies.
Protocol of Biuret Protein Assay
The protocol for performing the biuret protein assay is as follows:
Materials:
- Biuret reagent
- Protein sample
- Standard protein solution
- Spectrophotometer
- Cuvettes
- Distilled water
Procedure:
- Prepare the biuret reagent according to the manufacturer's instructions. Briefly, the biuret reagent can be prepared by dissolving 1 g of copper sulfate pentahydrate and 4 g of sodium potassium tartrate tetrahydrate in 100 mL of distilled water. Then, add 4 g of sodium hydroxide pellets and dissolve by stirring. Finally, bring the volume to 200 mL with distilled water.
- Prepare a standard protein solution of known concentration. You can use a standard protein such as bovine serum albumin (BSA). Prepare a stock solution of BSA by dissolving a known amount (e.g. 1 mg) in 1 mL of distilled water. Then, prepare a series of dilutions of the BSA stock solution to create a standard curve. For example, you can prepare 5 standard solutions with concentrations of 0.1, 0.2, 0.4, 0.6, and 0.8 mg/mL.
- Dilute the protein sample to an appropriate concentration using distilled water. The concentration of the protein sample should be within the linear range of the standard curve.
- Add 2 mL of biuret reagent to each sample and standard in a cuvette. Mix well by inverting the cuvette several times. It is important to mix the sample thoroughly with the biuret reagent to ensure complete reaction between the copper ions and the protein.
- Incubate the cuvettes at room temperature for 30 minutes. During this time, the biuret reagent reacts with the peptide bonds in the protein, forming a purple-colored complex. The intensity of the purple color is directly proportional to the protein concentration in the sample.
- Measure the absorbance of the samples and standard at 540 nm using a spectrophotometer. Set the spectrophotometer to zero using a blank cuvette filled with distilled water. Then, measure the absorbance of the standard solutions and the protein sample. It is important to measure the absorbance within a few minutes of adding the biuret reagent to the samples, as the color intensity may fade over time.
- Calculate the protein concentration of the samples by comparing the absorbance values to the standard curve obtained from the standard protein solutions. Plot the absorbance values of the standard solutions against their corresponding protein concentrations to create a standard curve. Use the standard curve to determine the protein concentration of the sample based on its absorbance value.
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