KA1: laboratory techniques for biologists: separation techniques Flashcards
describe the use of centrifugation to seperate substances of differeing denisty
-more dense components settle in the pellet
-less dense comp remain in the supernatant
what can paper and thin layer chromatography be used for?
separating different substances such as amino acids and sugars
what does the speed that each solute travels along the chromatogram depend on?
its differing solubility in the solvent
what is the main use of affinity chromatography?
separating proteins
how is a solid matrix or gel column created? (for use in affinity chromatography)
with specific molecules bound to the matrix or gel
describe the process of the solvent moving up the chromatography paper (in affinity chromatography)
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 the main use of gel electrophoresis?
separating proteins and nucleic acids
describe gel electrophoresis
charged macromolecules move through an electric field applied to a gel matrix.
how do native gels separate proteins?
shape, size and charge
why do native gels separate proteins by shape, size and charge?
as they do not denature the molecule
what does SDS-PAGE separate proteins by?
size alone
describe SDS-PAGE
gives all the molecules an equally negative charge and denatures them (therefore separating by size alone)
how can proteins be separated from a mixture?
using their isoelectric points (IEPs)
what is IEP?
the pH at which a soluble protein has no net charge and will precipitate out of solution
what happens if the solution is buffered to a specific pH?
only the protein(s) that have an IEP of that pH will precipitate
