Protein sequencing fragmenting/purification

carboxypeptidase A

-from C terminal

-ANY AA except R and K (and not if proline is R_n-1)

carboxypeptidase B

-from C terminal

-K & R (but not if proline is R_n-1)

cyanogen bromide

-from C terminal

-only M (and not if proline is R_n-1)

trypsin

-from C terminal

-K & R(but not if proline is R_n-1)

chymotrypsin

-from C terminal

-F, Y, W (but not if proline is R_n-1)

elastace

-from C terminal

-E, A, S, V (but not if proline is R_n-1)

salt fraction

-ammonium sulfate

-low salt, salt in, cut to remove unwanted proteins

-high salt, salt out, cut to precip out desired protein

column chromatography

-based on size, charge, polarity, and affinity to bind

size exclusionary chromatography

AKA GEL FILTRATION

-separates based on size

-larges elutes first, smallest last

ion-exchange chromatography

-anionic matrix= cation exchange (negative out first, then positive last)

-cationic matrix= anion exchange (positive out first, then negative last)

-based on charge

affinity chromatography

1 STEP CHROMATOGRAPHY

-ligand that binds to desired protein is attached to matrix, everything else washes out, then desired protein is recovered

dialysis

-separates molecules via size through equilibrium concentrations, removes smaller undesired compounds

electrophoresis

-analytical tool

-separates by molar weight/pI

-the farther down the smaller the protein, the more negative the charge

SDS-PAGE

-sodium dodecyl sulfate used to denature proteins to make them all the same shape (1 between every 2 AA)

-separated ONLY on molecular weight, smallest on bottom largest on top

isoelectric focusing

-uses pH gradient

-proteins migrate to where pH=pI

-largest pI at TOP, smallest pI at bottom

2D electrophoresis

-isoelectric focusing + SDS-PAGE

-separates based on pI and MW

-smaller MW lower down, smaller pI to the right!