Lecture 2b - protein techniques Flashcards
Proteome
the set of proteins expressed by a call under a particular set of conditions
Differential centrifugation
Spinning fractions at different speeds to separate out different components:
- nuclear
- mitochondria
- microsomal
Separation techniques
- salting out
- gel filtration chromatography
- ion exchange chromatography
- affinity chromatography
- Polyacrylamide gel electrophoreses (PAGE)
- SDS-PAGE
- Isoelectric focusing
- Density gradient centrifugation
- Immunoprecipitation
Gel filtration chromatography
Uses beads of a specific size that will slow proteins. The largest proteins will fall the fastest.
Ion exchange chromatography
Uses beads that are either positive or negative charged. Will attract protein of opposite charge. A buffer of different pH or a salt is used to remove proteins.
Affinity chromatography
Attaches a ligand to the bead for the protein to bind to. Knock it off by an excess of free glucose
Gel electrophoresis
Moves protein according to the charge. The gel is made of a cross-linked (think cheesecloth) polyacrylamide and placed vertically. Migrates down to the positive end.
SDS
Makes all the proteins of equal charge to mass ration, so proteins will only move according to mass.
Isoelectric focusing
Proteins are placed in a horizontal gel with a pH gradient. They will migrate to the point at which they have no net charge.
Density gradient centrifugation
After creating a solution of various densities, spinning will cause separation of particles based on density gradient.
Immunoprecipitation
The antibody bound to antigen will be heavier and will precipitate out, allowing it to be isolated
Tandem mass spectrography
Sample separated by ionization, then broken apart again.
