i. Introduction

Western blotting is an immunoblotting technique. Immunoblotting techniques rely on the specificity of binding between a molecule of interest and a probe to allow detection of the molecule of interest in a mixture of many other similar molecules. In Western blotting, the molecule of interest is a protein and the probe is typically an antibody raised against that particular protein.

The SDS PAGE technique is a prerequisite for Western blotting. Your goal is to use the Western blotting technique to determine if a specific protein (called GT50) is present in the cell lysate samples from the SDS PAGE module.

The sections in the Western Blotting module are:

ii. Transfer Step - The proteins in the SDS PAGE gel are transferred to a thin membrane using electrophoresis.

iii. Blocking and Additon of Antibodies - The membrane is treated with three different solutions. The first is a ‘blocking’ solution, the second is a primary antibody solution, and the final one is a secondary antibody solution. The secondary antibody has been modified to allow its to be visualized.

iv. Visualization - The membrane is washed in a solution that reacts (with the secondary antibody) to show where the protein of interest (if present) is bound to the membrane.