Chemistry
How SDS-PAGE Separates Proteins by Molecular Weight
Quick fact
SDS denatures proteins and gives every protein roughly the same negative charge per unit mass, so the only difference in how they move through the gel is their size.
Why this is interesting
Imagine being able to separate a mixture of proteins into neat bands, each one marking a different size. That’s exactly what SDS-PAGE does—but how does it make proteins move solely by their weight?
Read the full explanation
Understanding How SDS-PAGE Separates Proteins by Molecular Weight
To understand SDS-PAGE, think of a race through a forest of tangled roots. The runners are proteins, and the forest is a polyacrylamide gel—a mesh of cross-linked polymers. First, proteins are treated with SDS (sodium dodecyl sulfate), a detergent that unfolds them and coats them with negative charges. This makes all proteins rod-like with a similar charge-to-mass ratio. When an electric field is applied, proteins are pulled toward the positive electrode. As they move, they thread through the gel's pores. Smaller proteins slip through easily and run faster, while larger ones get tangled and move slower. After a set time, the proteins have separated into bands, with the smallest at the bottom. Comparing the positions of these bands to those of known-size markers reveals each protein's molecular weight.
A deeper explanation
The power of SDS-PAGE lies in its ability to cancel out differences in intrinsic charge and shape. SDS binds to proteins at a constant ratio (about 1.4 grams of SDS per gram of protein), giving each protein a uniform negative charge density. The disulfide bonds that hold protein structure together are often reduced too, ensuring full unfolding. In the gel, the pore size is determined by the concentration of acrylamide; higher percentages create smaller pores, optimal for separating smaller proteins. Under the electric field, the mobility of a protein is inversely proportional to the logarithm of its molecular weight, meaning that a plot of log(MW) versus migration distance produces a straight line. This allows scientists to estimate molecular weights with remarkable precision. SDS-PAGE is fundamental to biochemistry—it is used to assess protein purity, compare expression levels, and prepare samples for Western blotting or mass spectrometry.