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 SDS-PAGE gel, has expired (and is no longer able to denature). What is the role of SDS? What is the expected result, assuming these proteins interact with one another

Answer 1

SDS (sodium dodecyl sulfate) is a detergent, which is used for the isolation and identification of the proteins. It is used in the PAGE (polyacrylamide gel electrophoresis) process for the determination of the molecular weight of the protein. The SDS-PAGE isolates the proteins according to their mass because SDS is an ionic detergent denatures the protein and get binds to the protein to make them negatively charged uniformly. Thus when the current is applied, the bound proteins will migrate from the gel towards the positive electrode.

SDS (anionic detergent) disrupts the non-covalent bonding or interactions in the native proteins. If the expired SDS is used for the protein sample then it will not disrupt the non-covalent interaction between the proteins and the protein will not denature. The addition of expired or old SDS to the protein sample will not cause the adsorption of the dodecyl sulfate at the interface of the protein layers and the interface does not expose to the water (hydrophobic). Due to this protein will remain in a condensed form or may show turbidity.