lab 34 peptides and proteins

lab 34 peptides and proteins

lab 34 of course is one of the most common experiments performed by biochemistry students in colleges of science and technology. in this lab, students will learn how to use a spectrophotometer to study the properties and behavior of different kinds of peptides to better understand the basic concepts and techniques of biochemistry.

objectives

1. Familiarize with common peptides and proteins

2. Understand the basic principles and methods of spectrophotometry

3. Investigate the physical and chemical properties of peptides and proteins

4. Explore the relationship between structure and bioactivity

Materials

UV-Vis Spectrophotometer

Peptides and proteins

Acidic buffer solutions of different pH values

Sucrose standards

Phosphate buffer

Reagents for determining amino acid composition

Electrophoresis apparatus and gel electrophoresis reagents

Chlorophyll extraction solvent

Centrifuge

Distilled water

The experiments of this lab are mainly completed manually. In the process of using the spectral photometer to determine the absorbance of peptide standard solutions of different concentrations, attention must be paid to controlling experimental variables such as the temperature and time of measurement to ensure data accuracy. To reduce experimental误差, appropriate measures such as multiple measurements and averaging are recommended.

experiment process

1. Preparation of peptides and proteins

First, prepare acidic buffer solutions of different pH values.
Using a 50 mM HCl solution, standard buffers for predicting the pH of peptides and determine the pH of the sample to be tested.
2. Preparation of sample solutions
Weigh the peptide samples and add them to appropriate amounts of buffer. Mix well and store at room temperature.
3. Determine the absorbance of standard solutions
Use a UV-Vis spectrophotometer to measure the absorbance of standard solutions of different concentrations and record the data.
4. Determine the absorbance of sample solutions
Using the same concentration of buffers for standard solutions as the sample solutions, measure the absorbance of sample solutions at room temperature.
5. Construct a calibration curve of calibration
Using the standard absorption values and standard concentrations of standard peptides to construct a calibration curve.
6. Determine the amino acid content in Peptides
Determine the amount of arginine, leucine, and alanine in peptides by comparing with standard amino acid solutions.

7. Use electrophoresis
If necessary, use electrophoresis to further study the purity and molecular weight distribution of peptides

8. Data analysis
Use software provided in the spectrophotometer to process and analyze experimental data

Preparation method

The procedures for detecting the absorption spectrum of the protein standard solution include dissolving the appropriate amount of protein in a suitable amount of buffer and then diluting with water to a desired concentration. It is important to ensure that the sample is freshly prepared before measurement every day. The protein concentration standard used to prepare the standard solution should be purchased from professional biology supply companies.

Procedure for measuring the absorbance of standard solutions

1. Open the spectrophotometer and set the wavelength to be measured.
2. Place the sample cuvette into the spectrophotometer and measure the initial absorbance of the standard solution blank.
3. Add the standard solution to a designated cuvette in the spectrophotometer and record the absorbance of each standard solution.
4. Repeat steps 3 and 4 multiple times to get an average absorbance value for each standard solution.
5. Calculate the concentration of the standard solution based on the slope of the plot of absorbance vs concentration.

Procedure for measuring the absorbance of sample solutions

1. Follow the steps from steps 1 to 4 for preparing standard solutions as described above.
2. When adding the sample solution to the cuvette, be careful not to stir it too much to avoid mixing errors.
3. Record the absorbance of the sample solution, making sure to include the reference absorbance reading.
4. Repeat steps 1 to 3 for each sample solution.

Data analysis

After obtaining absorbance data, use the software provided in the spectrophotometer to process and analyze experimental data. This typically involves fitting a curve to the absorbance versus concentration data, as in Equation (1). The absorbance of the sample solution at concentration (A) can be calculated from the absorbance of the standard solution (A) using Equation (2) below: