Question

A graduate student has performed an in vitro pull-down assay to determine whether two proteins, Protein A and Protein B, interact with each other. Protein A is known to be 35 kDa and Protein B is 60 kDa. To determine whether there is an interaction the graduate student runs the sample down an SDS-PAGE gel, which is later stained with instablue dye. Unfortunately, the SDS, found in both the lysis buffer and SDSPAGE gel, has expired (and is no longer able to denature). What is the role of SDS (1)? What is the expected result, assuming these proteins interact with one another?

Answer 1

SDS is a detergent that is used to denature proteins. It can denature polyacrylamide gel electrophoresis for the determination of protein molecular weight.

The pull-down assay is an in vitro method used to determine a physical interaction between two or more proteins. Pull-down assay is a form of affinity purification. This process involves isolation of a protein complex by adsorbing the complex onto beads. In beads there are immobilized ligands that bind specifically to a component of the complex. If there is protein-protein interaction, the complex will bind to the immobilized ligands via an affinity tag and then you can analyze the positive result.