Unit 1.1.c Flashcards
What is the use of a centrifuge?
To separate substances of differing density
More dense components settle in the pellet; less dense components remain in the supernatant.
What can paper and thin chromatography be used for
For separating different substances such as amino acids and sugars
The speed that each solute travels along the chromatogram depends on its differing solubility in the solvent used.
What is affinity chromatography used for?
Separating proteins
What is the principle of affinity chromatography
A solid matrix or gel column is created with specific molecules bound to the matrix or gel. Soluble, target proteins in a mixture, with a high affinity for these molecules, become attached to them as the mixture passes down the column. Other non-target molecules with a weaker affinity are washed out
What is gel electrophoresis used for?
Separating proteins and nucleic acids
What is the principle of gel electrophoresis?
Charged macromolecules move through an electric field applied to a gel matrix.
How do native gels separate proteins?
Native gels do not denature the molecule so that separation is by shape, size and charge
How does SDS-PAGE separate proteins?
SDS–PAGE gives all the molecules an equally negative charge and denatures them, separating proteins by size alone.
What is IEP and what is it used for?
IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.
Proteins can be separated from a mixture using their isoelectric points (IEPs)
What happens if the solution is buffered to a specific pH?
only the protein(s) that have an IEP of that pH will precipitate
How can proteins be separated using their IEPs in electrophoresis?
Soluble proteins can be separated using an electric field and a pH gradient. A protein stops migrating through the gel at its IEP in the pH gradient because it has no net charge.
