What is a better way of determining when the solution milk goes clear in trypsin experiment?

Determining the endpoint of a trypsin casein hydrolysis experiment by observing when the solution turns clear can be subjective and imprecise. A more quantitative and accurate method is to use spectrophotometer to measure the absorbance of the solution at a specific wavelength.

Here's an improved procedure for determining the endpoint of the trypsin experiment using a spectrophotometer:

Materials:

- Trypsin solution

- Casein substrate solution

- Sodium hydroxide (NaOH) solution (0.1 M)

- Spectrophotometer

- Cuvettes

Procedure:

1. Prepare the Reaction Mixture:

- In a cuvette, mix a specific volume of casein substrate solution (e.g., 1 mL) with an appropriate volume of trypsin solution (e.g., 0.1 mL).

- Incubate the mixture at a suitable temperature (e.g., 37°C) for a desired time (e.g., several minutes).

2. Stop the Reaction:

- At specific time intervals (e.g., every minute), withdraw a small volume of the reaction mixture (e.g., 0.1 mL) and transfer it to a separate cuvette.

- Immediately add a sufficient volume of sodium hydroxide solution (e.g., 1 mL) to stop the reaction.

3. Measure Absorbance:

- Use the spectrophotometer to measure the absorbance of each stopped reaction mixture at a specific wavelength (e.g., 440 nm).

4. Plot the Absorbance:

- Plot a graph with time (x-axis) and absorbance (y-axis).

5. Determine the Endpoint:

- Analyze the absorbance values over time. The endpoint is the time point at which the absorbance reaches a plateau or shows a significant decrease, indicating the extensive hydrolysis of casein.

This method provides a more objective and quantitative endpoint determination compared to visually observing the clearing of the solution, as it relies on measuring the change in absorbance due to the release of smaller peptides and amino acids during casein hydrolysis.