Unit 1 - Topic 1 - Laboratory Techniques for Biologists - Section C - Separation techniques Flashcards
What is a centrifuge used for?
Centrifuge are used to separate substances of differing density
What are the names for the separated components?
More dense components settle in the pellet; less dense components remain in the supernatant.
What can be used for separating different substances such as amino acids and sugars?
Paper and thin layer chromatography
What does solubility in the solvent effect?
The speed that each solute travels along the chromatogram
Explain affinity chromatography and its use in separating proteins
- 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 does gel electrophoresis separate?
Proteins and nucleic acids
How does gel electrophoresis work
Charged macromolecules move though an electric field applied to a gel matrix
What do native gels separate proteins by
Shape, size and charge
What do native gels separate proteins by and how do they do this
Shape, size and charge. They do not denature the molecules
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 an IEP and what does it separate
The isoelectric point separates proteins. An IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.
How does a buffer effect IEP
If the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate
Proteins can also be separated using their IEPs in electrophoresis how is this done
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.
