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MODULE 2: PROTEIN PURIFICATION & CHARACTERIZATION - Coggle Diagram
MODULE 2: PROTEIN PURIFICATION & CHARACTERIZATION
Separative techniques
gel filtration (size-exclusion)
bigger molecules are excluded from the pores --> elute first
ion-exchange chromatography
ex. positively charged wall
add sample --> all the anions will stick to the wall and the cations will elute out --> add buffer with strong ionic concentration to elute all the strongly charged anions
centrifugation
density gradient
relatively impure fractions (the pellets) from differential centrifugations are layered on top of a solution containing gradient of a dense, nonionic solute
particles have the same mass but different density
collect samples by either
sucking each sample from the top of the tube
or poke a hole at the bottom and collect samples
differential: separation of cellular organelles and subcellular particles based on sedimentary profile (bigger particles will sediment faster)
successive centrifugation at increasing G forces (imposed by increasing rotary speeds)
after 1st centrifugation: get pellet and supernatant 1 (the big stuff sediments first --> pellet)
pour supernatant 1 into tube and repeat centrifugation
affinity chromatography
only molecules with the right binding properties to the affinity ligands on the walls of the column will bind --> everything else will be washed ouuut with buffer
elute the target molecules by adding an elution buffer of stronger pH
FPLC and HPLC
SDS-PAGE (based on m/z)
proteins bind to the negatively charged SDS molecules --> the protein-SDS complexes move down and the molecules with the smallest mass/charge ratio will move farther down the column
SDS can either be VERY cross-linked (good for small molecules) or not tightly cross-linked (can allow bigger molecules to move through)
isoelectric focusing (based on pI)
pH (low acidic pH's are associated with positive charge and high basic pH's are associated with negative charge charge)
apply voltage --> molecules move based on their isoelectric points
2D electrophoresis: combines SDS-PAGE and IEF
Purity
specific activity: number of enzyme units per mg protein
unit of enzyme activity: amount of enzyme capable of transforming 1.0 micro mol of substrate to product per minute
Protein sequences
Edman degradation
In mild alkaline conditions: Edman's reagent (Phenyl isothiocyanate) labels the N terminal amino acid --> the labeled amino acid is a phenylthiocarbamoyl (PTC) derivative
under heat and acidic conditions, PTC derivative is cleaved from the peptide and extracted by organic solvents/acidified --> creates phenylthiohydantoin (PTH) amino acid derivative (the PTH derivative can be found using HPLC)
problem w Edman degradation: the N-terminal can be blocked
End group analysis
Sanger's reagent (DNF) and dansyl chloride reacts w the N-terminal amino acid
site specific proteolysis
cyanogen bromide --> carboxyl side of Met
trypsin --> carboxyl side of Lys and Arg
Thrombin --> carboxyl side of Arg
Chymotrypsin --> carboxyl side of trp, tyr, phe, met, leu