Protein Isolation & Analysis Techniques Flashcards
3 kinds of electrophoresis
Native PAGE, SDS PAGE, Isoelectric focusing
Native PAGE
- maintains the protein’s shape
- proteins separated in electric field on basis of net negative charge/size
- smaller and more negatively charged proteins migrate faster through gel
- results difficult to compare because mass-to-charge ratio differs for each protein
SDS-PAGE
- used to determine degree of purity, molecular mass, and number of polypeptide subunits in protein
- sample is treated with reducing agent to break disulfide bonds and then SDS (an anionic detergent) denatures and covers proteins with an overall negative charge
- proteins all have identical mass-to-charge ratio after treatment with SDS, thus they are separated on the basis of their mass
- smallest proteins move farthest
Isoelectric focusing
- proteins separated by their pI
- pH gradient applied to gel
- each protein migrates through gel until it reaches the point where it has no net charge (its pI)
Chromatography
separates protein mixtures on the basis of their affinity for a stationary or mobile phase
column chromatography
uses beads of a polar compound as stationary phase with nonpolar solvent as mobile phase
ion-exchange chromatography
- proteins separated on basis of their net charge
- uses charged column and variably saline eluent
- anion exchange and cation exchange
anion exchange chromatography
column contains positive beads to which negatively charged proteins bind
cation exchange chromatography
column contains negatively charged beads to which positively charged proteins bind
size exclusion chromatography
- relies on porous beads
- larger molecules elute first because they are not trapped in small pores
affinity chromatography
uses a bound receptor or ligand and an eluent with a free ligand or a receptor for the protein of interest
Protein structure is primarily determined through:
X-ray crystallography… although NMR can also be used
