Question

Explain what you must do to properly denature a protein for a gel. What chemicals are used and what is their specific purpose?

Answer 1

Answer: Proteins are the polymers of amino acids, the polypeptides which have secondary/tertiary structure. To separate proteins based on their size they are run on a gel under the influence of current, proteins with smaller size will run ahead and with larger sizes will lag behind.

Before subjecting to electrophoresis, proteins need to be denatured, as the process of denaturation will linearise them so that their structural complexity will not interfere with their separation on the gel.

The process of denaturation consists of treatment with SDS (sodium dodocyl sulphate), a reducing agent and heating.

SDS is a detergent with hydrophobic hydrocarbon tail, attached to an ionic sulphate group. When SDS interacts with protein, its hydrophobic tail dissolves hydrophobic region of the protein and the sulphate breaks ionic bonds. By this protein loses its secondary and tertiary structure.

A reducing agent like mercaptoethanol (or dithiothreitol) breaks the disulfide linkages and helps in linearising the protein molecule.

The protein sample is boiled in the loading buffer containing Tris-HCl, SDS, bromophenol blue(tracking dye), glycerol(increases the density of the sample than the buffer so that the sample remains at the bottom of the well), EDTA (ethylene diamine tetraacetic acid acts as a chelating agent for divalent cations like Ca and Mg and reduces the proteolytic activity of the enzymes) heating helps in complete denaturation of the protein. As the protein might be bound to certain lipids through hydrophobic interactions, heating dissociates these interactions and exposes hydrophobic ends for denaturation by SDS.