Laboratory Techniques For Biologists: Separation Techniques

Question 1

Centrifugation

Answer 1

Centrifugation is a method for separating materials in suspension according to their differing density.

The more dense components form a pellet at the bottom of the tube, whereas the less dense liquid fraction above is named the supernatant.

Question 2

Chromatography

Answer 2

Paper and thin-layer chromatography -

Can be sued 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.

Affinity chromatography -

Affinity chromatogrpahy is used for the separation of one specific soluble protein from a mixture.
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.

Question 3

Gel Electrophoresis

Answer 3

Gel electrophoresis uses a current flowing through a buffer to separate either proteins or nucleic acids.

• Size and charge are factors that affect the rate at which any macromolecule migrates through a gel

Charged macromolecules move through an electric field applied to a gel matrix.

Native gels do not denature the molecule so that separation is by shape, size and charge.

SDS-PAGE separates proteins by size alone

SDS–PAGE gives all the molecules an equally negative charge and denatures them, separating proteins by size alone.

Question 4

Isoelectric point
(IEP)

Answer 4

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)

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

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.