Unit 1 Part 1c (Separation Techniques)

Question 1

How does centrifugation separates substances?

Answer 1

Separates by differing densities
More dense components settle in the pellet; less dense components remain in the supernatent

Question 2

How does paper and thin layer chromatography separate substances?

Answer 2

Separates 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

Question 3

How does affinity chromatography separate substances?

Answer 3

Separates 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 they pass down the column. Other non-target molecules with a weaker affinity are washed out.

Question 4

How does gel electrophoresis separate substances?

Answer 4

Separates proteins and nucleic acids
Charged macromolecules move through an electric field applied to a gel matrix

Question 5

How do native gels separate substances?

Answer 5

Does not denature the molecule so can separate proteins by their shape, size and charge

Question 6

How does SDS-PAGE separate substances?

Answer 6

Separates by size alone
Gives all molecules an equally negative charge and denatures them

Question 7

How does separation by isoelectric points work?

Answer 7

IEP: the pH at which a soluble protein has no net charge and will precipitate out of the solution.
If solution is buffered to a specific pH, only the proteins that have an IEP of that pH will precipitate

Question 8

How can separation by IEP be applied to electrophoresis?

Answer 8

A protein stop migrating through the gel at its IEP in the oH gradient because it has no net charge