Biochemical techniques

• Electrophoresis is the movement of charged molecules in an electric field, usually performed on a gel such as Agarose or Polyacrylamide.
• Agarose gels are polysaccharide derived from seaweed, form a hydrated gel when cross-links reform on cooling, and are used for DNA.
• Electrophoresis of DNA depends on the size, conformation, and linear, circular, or supercoiled state of the DNA.
• Staining for DNA uses intercalating dyes such as ethidium bromide, which fit between stacked bases and have a fluorescence intensity that increases on binding to DNA.
• Acrylamide gels are a copolymer of acrylamide and bisacrylamide, form a gel in water, and are used for polypeptides.
• Polyacrylamide gels are cross-linked polyacrylamide gels, formed by acrylamide monomer polymerisation in the presence of bisacrylamide, and are used for proteins.
• The vertical gel system consists of a lower tank (anode), upper tank (cathode), glass plates, and spacers.
• Staining for protein can be done with Coomassie (brilliant) blue, which is commonly used but has low sensitivity, or with Silver, which has high sensitivity.
• Column chromatography involves packing the stationary phase into a glass column and applying a mixture of analytes.
• In gas (liquid) chromatography, the stationary phase consists of a high-boiling-point liquid silicone grease or wax coated onto the internal wall of the column.
• The Rf value can be used to identify compounds due to their uniqueness to each compound.
• Gas (liquid) chromatography (GC) involves a mobile phase that is an inert gas carrying a vaporized sample, and a stationary phase that is liquid adsorbed on a solid matrix.
• Affinity chromatography exploits the unique property of extremely specific biological interactions, resulting in separation and purification.
• The stationary phase in column chromatography is coated onto discrete small particles (matrix) and packed into the column or applied as a thin film to the inside wall of the column.
• Size exclusion chromatography involves molecular sieving/molecular exclusion chromatography, where larger molecules are excluded and move rapidly through the column, and smaller molecules are distributed between the mobile phase inside and outside the particles and pass through the column slower.
• Enzyme purification involves binding to a specific ligand and recovery by displacement.
• In column chromatography, the mobile phase (eluent) is passed through the column using a pumping system or applied gas pressure.