What are the components in the sample loading buffer?
Posted May 2, 2023
A sample loading buffer is made up of five main components:
- SDS - SDS performs two important functions. It denatures proteins by disrupting non-covalent bonds, destabilizing the secondary, tertiary, and quaternary structural assemblies. Secondly, it also ensures that all protein analytes migrate in the same direction during an SDS-PAGE assay by imparting a net negative charge on all the protein analytes.
- Reducing Agent - The reducing agent helps to disrupt covalent disulfide bonds that are unaffected by SDS.
- Tris - Tris is responsible for keeping the Laemmli buffer chemically stable by maintaining a pH of 6.8, which helps achieve maximum resolution for SDS-PAGE experiments. Another function of Tris is preventing proteases from degrading the analytes by inhibiting the number of enzymes.
- Glycerol - Glycerol prevents the analytes from diffusing into the gel tank. This is because glycerol is denser than water. When sufficient glycerol is mixed into the Laemmli buffer-sample mixture, it makes the whole solution denser than water and causes it to sink when loaded onto the SDS-Page gel. This prevents the analytes from diffusing into the gel tank and allows you to proceed with your experiment.
- Dye - Adding a dye stains the Laemmli buffer, making it easier to identify the samples when they are loaded onto the SDS-PAGE gel.