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SDS-PAGE is a molecular weight-based technique that separates protein mixtures by size. Proteins are treated with SDS, a negatively charged detergent that denatures them and adds uniform negative charge, and β-mercaptoethanol, a reducing agent that breaks disulfide bonds. When loaded onto a polyacrylamide gel under voltage, smaller proteins migrate faster through the gel's pores, achieving size-based separation.

The stacking gel has large pores due to low acrylamide concentration and high electrical resistance, allowing proteins to stack like coins before entering the resolving gel simultaneously. The resolving gel has smaller pores from higher acrylamide concentration, acting as a molecular sieve to separate proteins by size. This two-gel system ensures uniform entry and precise separation.

β-mercaptoethanol is a reducing agent that breaks disulfide bonds linking protein chains together. This treatment unfolds and linearizes proteins, ensuring they migrate through the gel based solely on molecular weight rather than their native three-dimensional structure, enabling accurate size-based separation.

Pore size is determined during gel preparation by adjusting the concentration of acrylamide monomers and bisacrylamide, the cross-linking agent. Higher acrylamide concentration produces smaller pores, while lower concentration creates larger pores. Ammonium persulfate catalyzes the polymerization reaction that forms the polyacrylamide gel.

The sample buffer contains SDS, β-mercaptoethanol, glycerol, and bromophenol blue. Glycerol increases sample density to keep it at the well bottom, preventing overflow into the buffer. Bromophenol blue serves as a tracking dye, indicating protein progress through the gel during electrophoresis.

Separated protein bands are commonly visualized using Coomassie blue staining, which binds to proteins and produces colored bands. The molecular weight of each band is determined by comparing its migration distance to reference marker proteins of known sizes, allowing quantitative analysis of protein composition.

SDS-PAGE cannot provide information about enzyme activity, cofactors, or protein binding interactions since it only separates by size. Additionally, it is challenging to analyze highly acidic or basic proteins using this technique. For more detailed protein characterization, complementary methods like peptide identification using tandem mass spectrometry may be needed.