Title: Learn: SDS-PAGE
Course: BTEC-2020-301-302-303-F21 > Section: Protein Gels

Module2 SDS-PAGE

SDS-PAGE (Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis) is a technique that uses SDS, heat, and DTT to drastically reduce the effects of proteins’ variable charge and shape, so that molecular weight determines migration. SDS-PAGE is a type of denaturing gel electrophoresis

SDS-PAGE is often used to determine a protein’s molecular weight, or to monitor a protein’s abundance in cells or solutions. In this course, you will use it to monitor the expression of an enzyme in E. coli, and to assess its abundance and purity through the purification process.

Unlike in DNA gels where you know that mass is the only factor determining migration, in a nondenaturing protein gel there are three variables that can influence migration: mass, shape, and charge. SDS-PAGE is a technique that allows us to (mostly) isolate a single variable: mass.

SDS-PAGE: Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis.

Sodium Dodecyl Sulfate (SDS) is also known as Sodium Lauryl Sulfate and is the main detergent in most shampoos. When you use shampoo, the detergent surrounds particles of dirt and oil, making it easy to rinse them away.

In SDS-PAGE, the ability of SDS to coat proteins is critical. The goal isn’t to wash the proteins away, but to make them more uniform in terms of charge and shape. SDS is negatively charged, and binds to most proteins in a fixed ratio: 1.4 g of SDS per g of protein. By adding a large amount of negative charge to each protein, the native charge of the protein is effectively overwhelmed; all SDS-treated proteins have a negative charge that is approximately proportional to the mass of the protein. In the process of coating proteins, SDS disrupts structure arising from native charges and hydrophobicity, causing most proteins to take on a rod-like shape. Thus, in addition to making all proteins negatively charged, SDS also makes them all roughly the same shape.

Denaturation of a protein (bottom-left) by sodium dodecyl sulfate (top-left). Detergent molecules coat the unfolded protein chain, which adopts a rod-like shape as a consequence of electrostatic repulsion. Negative charges contributed by SDS overwhelm the native charge of the protein.
By Fdardel (Own work) [CC BY-SA 4.0], via Wikimedia Commons

To summarize, SDS effects four important changes to our proteins:

- Overwhelms the native charge
- Applies a negative charge proportional to the protein’s mass
- Denatures native structure
- Makes most proteins become rod-shaped