Protein Isolation Flashcards
Electrophoresis
uses a gel matrix to observe the migration of proteins in response to an electric field
Native PAGE
maintains the proteins shape but results are difficult to compare because the mass-to-charge ratio differs for each protein
SDS-PAGE
denatures the proteins and masks the native charge so that comparison of size is more accurate but the functional protein cannot be recaptured from the gel
Isoelectric Focusing
separates proteins by their isoelectric point (pI); the protein migrates toward na electrode until it reaches a region of the gel where pH = pI of the protein
Chromatography
separates protein mixtures on the basis of their affinity for a stationary phase or a mobile phase
Column chromatography
uses beads of a polar compound, like silica or alumina (stationary phase), with a non polar solvent (mobile phase)
Ion-exchange chromatography
uses a charged column and a variably saline eluent
Size exclusion chromatography
relies on porous beads
Larger molecules elute first because that are not trapped in the small pores
Affinity chromatography
uses a bound receptor or ligand and an fluent with free ligand or a receptor for the protein of interest
Homogenization
crushing, grinding, or blending the tissue of interest into an evenly mixed solution
Centrifugation
isolates the proteins from much smaller molecules before other isolation techniques must be employed
SDS detergent disrupts
all noncovalent interactions
retention time
the amount of time a solute remains in the stationary phase
what can be triggered to help elute the protein of interest in a column chromatography?
solvent polarity
pH
salinity
2 drawbacks of using the affinity chromatography
- the protein of interest may not elute from the column because its affinity is too high
- it may be permanently bound to the free receptor in the eluent
