1.c. Separation Techniques Flashcards
What is centrifugation used to seperate?
Substances of differing density
In centrifugation where will more dense compounds settle?
The pellet
In centrifugation where less dense compounds settle?
The supernatant
What can Paper and Thin-layer chromatography be used for?
Separating different substances such as amino acids and sugars
What factors determines the speed at which each solvent travels at along the chromatogram?
Differing solubility in the solvent used.
How does affinity chromatography work?
- 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 through an electrical field applied to a gel matrix
What do Native gels separate and how?
Separate proteins by their shape, size and charge (does not denature the molecules and allows them to do this).
What do SDS-PAGE separate and how?
Separates proteins by size alone by giving all molecules an equally negative charge and denatures them
How can proteins be separated from a mixture?
Using their Isoelectric points (IEP)
What are IEPs?
The pH at which a soluble protein has no net charge and will precipitate out of a solution
What happens to proteins if the pH is buffered?
Only the proteins at that have an IEP of that pH will precipitate
How else can proteins be separated using their IEPs?
Using electrphoresis
