PRAKTIKUM BIOKIMIA - PENENTUAN KADAR PROTEIN DENGAN METODE BIURET

Summary

This video demonstrates the Biuret method for determining protein concentration in a sample. It provides a detailed step-by-step guide on sample preparation, including cutting and grinding the sample, centrifugation, and the creation of protein standard solutions with varying concentrations. The procedure includes mixing the samples with Biuret reagent, incubating, and measuring absorbance at 540 nm using a UV-Vis spectrophotometer. The protein content is then quantified by comparing the sample's absorbance to a standard curve. This method is widely used for protein analysis in biological studies.

Takeaways

• 😀 The experiment aims to determine the protein concentration in a sample using the Biuret method.
• 😀 Key materials for the experiment include a chemical beaker, test tubes, centrifuge, pipettes, and Biuret reagent.
• 😀 The protein sample, such as tilapia, is cut into small pieces, ground to a fine powder, and weighed before being processed.
• 😀 The protein sample is placed in a centrifuge tube with 10 mL of aquades before being centrifuged at 3500 RPM for 10 minutes.
• 😀 Standard protein solutions with concentrations ranging from 1 mg/mL to 5 mg/mL are prepared by diluting a 10 mg/mL stock solution.
• 😀 Biuret reagent is added to both the standard solutions and the protein sample, followed by mixing to ensure homogeneity.
• 😀 The reaction mixtures are incubated at 37°C for 10 minutes, then allowed to cool at room temperature for 3 minutes.
• 😀 Absorbance is measured using a UV spectrophotometer at a wavelength of 540 nm to assess protein concentration.
• 😀 A calibration curve is created using the absorbance values of the standard solutions to determine the protein concentration in the sample.
• 😀 The spectrophotometer's UV Perfect 2.3 22 software is used for measuring absorbance and saving results efficiently.
• 😀 Proper handling of equipment, such as ensuring balance in the centrifuge and using clean cuvettes, is crucial for accurate results.

Q & A

What is the main objective of the experiment?

The main objective of the experiment is to determine the protein content in a sample using the Biuret method.

What equipment is used in the experiment?

The equipment used includes a beaker, volumetric flask, mortar and pestle, centrifuge tubes, pipette, stirrer, graduated cylinder, test tube rack with test tubes, knife, digital balance, and centrifuge.

What are the key materials required for the experiment?

The key materials required include the protein sample (such as tilapia fish), distilled water (aquades), Biuret reagent, and albumin solution.

How is the sample prepared before the experiment?

The sample is first cut into small pieces, then ground into a fine powder using a mortar and pestle. Afterward, it is weighed (1 gram) using a digital balance.

How is the protein sample processed after weighing?

After weighing, the sample is placed in a centrifuge tube with 10 ml of distilled water. The tube is then centrifuged for 10 minutes at a speed of 3500 RPM.

What is the purpose of preparing standard solutions in the experiment?

Standard solutions are prepared to create different concentrations of protein (1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml) to compare the absorbance of the sample and determine its protein content.

How is the standard solution of 5 mg/ml prepared?

To prepare the 5 mg/ml standard solution, 5 ml of the 10 mg/ml stock protein solution is added to a 10 ml volumetric flask and then diluted with distilled water up to the mark.

What happens after the protein solutions are prepared?

After preparing the protein solutions, 1 ml of each standard solution is added to separate test tubes. 1 ml of distilled water is added to one tube as a blank, and 1 ml of the protein sample is added to another tube as the sample.

How are the test tubes treated after adding the solutions?

Each test tube is then added with 5 ml of Biuret reagent, mixed thoroughly, and incubated at 37°C for 10 minutes. After incubation, the tubes are left at room temperature for 3 minutes.

How is the absorbance of the sample and standard solutions measured?

The absorbance is measured using a UV-Vis spectrophotometer at a wavelength of 540 nm. The absorbance values for the blank, standard solutions, and sample are recorded, and the protein concentration of the sample is determined based on the standard curve.