Western Blot Diary 15th January 2025

Today we met up to finish our labs before the submission for our draft.

When we met in the lab we learned that this project is based on our independent motivation. So we have to follow the method that we had printed off

The main thing we did was to calculate the concentrations of the buffers. In addition, we went back to the library to figure out the next steps in regard to our electrophoresis dissertations

Calculating Buffer Calculations

We had to do a lot of calculations to determine the right concentrians to add into each buffer.

There are different formulas we had to use in order to calcualte ceratin concentrations

lamelli buffer is essential for maintaining protein stability during the electrophoresis process, and we calculated that we need to prepare 1X concentration for optimal results.

4% SDS = 0.4mL

10% Beta-Mercaptoethanol = 1mL

20% Glycerol = 2

0.01% Bromophenol Blue =

0.0125 M Tris-HCl =

pH 6.8

Running buffer is used for maintaining the pH and ionic strength during the electrophoresis, and we determined that a 1X Tris-Glycine running buffer is ideal to ensure effective protein migration.

Remember to add the pH indicator to the running buffer to monitor the pH levels throughout the experiment, ensuring that the conditions remain stable for accurate results. However the problem with using ph indicator paper is that reading the result accurately is more difficult as its a subjective reading instead of a quantitative measurement which could lead to data incosistancies.

We added NaOH into the running buffer to ensure that the pH was to our desired level of 8.3