Unit one essay one Flashcards
(1) Centrifugation: Separation by size and density
Centrifugation separates substances by size and density, with the densest materials forming a pellet at the bottom and the remaining liquid called the supernatant.
Chromatography Overview: Separation by solubility using mobile and stationary phases
Chromatography separates mixtures (proteins, amino acids, sugars) by solubility. A mobile phase dissolves the mixture and carries it through a stationary phase, with components traveling at different rates.
Paper Chromatography:
Paper: Hydrophilic paper (stationary) with hydrophobic solvent (mobile) separates based on solubility.
Thin Layer Chromatography:
Thin Layer: Silica gel or cellulose (stationary) and a solvent (mobile) move compounds up a plate, separating them based on solubility and adherence to the plate.
Affinity Chromatography:
Affinity: Separates proteins using a specific binding interaction between a ligand and protein. Ligands are immobilized in a column; proteins with high affinity bind to them and are later stripped off.
Gel Electrophoresis:
Gel Electrophoresis separates proteins and nucleic acids by size and charge using an electric current. The gel acts as a sieve, separating molecules based on their response to the electric field.
SDS-PAGE:
SDS-PAGE: Proteins are denatured, given a uniform negative charge, and separated by size alone, with smaller proteins traveling faster towards the positive electrode.
Isoelectric Point (IEP):
Isoelectric Point (IEP): Proteins carry different charges based on pH. At the IEP (no net charge), proteins are least soluble and precipitate out. A specific pH buffer can collect specific proteins based on their IEP.
Isoelectric Focusing:
Isoelectric Focusing: Proteins are separated by IEP using gel electrophoresis with a pH gradient. When proteins reach the region matching their IEP, they stop moving and form visible bands.
