Purification / Analysis Flashcards
separation by charge is called
ion-exchange chromatography
ion-exchange chromatography
cation and anion
cation has ( - ) charged beads… ( - ) proteins elute 1st
anion has ( + ) charged beads… ( + ) proteins elute 1st
ion-exchange chromatography solvent
salt solution at low [ ] to begin with
elute with high [ ] salt (competes with binding sites)
hydrophobic chromatography
start with high [ ] salt to encourage NP binding
solid phase is NP alkyl groups
lower salt [ ] to elute
polar proteins elute 1st
separation by size is called
size-exclusion
gel filtration
size exclusion/gel filtration
solid phase has beads with “mazes”
small proteins get trapped in “mazes” elute 2nd
large proteins bypass “mazes” elute 1st
binding-affinity
genetically engineer a binding affinity
to elute, add a lot of the binding affinity agent
goal of protein purification?
get a pure, functional folded protein
protein analysis goal
learn info (size) of a protein sample (pure or mix)
destroys protein in process
SDS-PAGE definition
sodium dodecyl sulfate - polyacrylamide gel electrophoresis
not used for purification
what happens in SDS-PAGE?
add SDS to unfold protein and add a ( - ) charge
deposit in gel and run electrophoresis
does the size of ( - ) charge in SDS-PAGE effect movement through gel?
no, it is balanced to mass ratio of protein
do smaller proteins move faster in SDS-PAGE?
yes, big proteins get caught in the gel
isoelectric focusing
separate based on pI (charge)
gel w/ pH gradient
often run in 2D gel
2D gel
run isoelectric focusing
flip 90 degrees then run SDS-PAGE
