Centrifugation and electrophoresis

Centrifugation spins samples at high speed so that particles settle according to their size and density, like how a washing machine's spin throws water outwards.

• Differential centrifugation: spinning at increasing speeds separates parts of a cell in turn: nuclei first, then mitochondria and chloroplasts.
• Density-gradient centrifugation: separates particles of similar size but different density, each settling at the layer matching its density.
• Ultracentrifugation: very high speeds, about 1,00,000 × g (gravity) or more.

Electrophoresis ("electro" = electricity, "phoresis" = movement) separates charged molecules by making them move in an electric field. How fast each moves depends on its charge compared to its mass. It was first seen in 1807, with clay particles in water.

Agarose gel electrophoresis separates DNA by size. DNA is negatively charged (because of its phosphate groups), so it moves towards the positive electrode (anode), at 80-120 volts, through a jelly-like gel. Smaller pieces slip through the gel faster and travel furthest. A DNA ladder (pieces of known size) run alongside shows the sizes. Ethidium bromide makes DNA glow under UV light, but it may cause cancer, so it must be handled with care.

SDS-PAGE separates proteins by size. The detergent SDS coats all proteins with negative charge, so they separate by size alone. The gel is acrylamide cross-linked with BIS. Looser gels (4-8%) suit large proteins; tighter gels (12-20%) suit small ones. Proteins are stained with Coomassie Brilliant Blue.

Tracking dyes (bromophenol blue, xylene cyanol, Orange G) show how far the run has gone. And a bit of history: agar, from red seaweed, became the standard medium for growing microbes when Fanny Hesse suggested it to Robert Koch's laboratory; agarose is purified from agar.