protein separation and purification Flashcards
protein property: solubility
method: disrupt cell membranes and create crude extract
protein property: solubility (2)
precipitation with ammonium sulfate (salting out)
protein property: size/shape
method: size exclusion chromatography
protein property: isoelectric point (charge)
method: ion exchange chromatography
protein property: binding to small molecules
method: affinity chromatography
sonicator
sound waves disrupt cell membranes
french press
pressure disrupt cell membrane
decant off supernatant with soluble protein produces what?
soluble lysate
at low salt concentrations, the solubility of a protein often increases
salting in
as the concentration of salt increases, the solubility of protein decreases until it precipitates
salting out
why do we use ammonium sulfate to precipitate proteins?
has a larger effect on solubility than other salts and it is very soluble so that solutions can be made at high concentrations
low [salt]
strong attractive force between proteins
optimal [salt]
salt ions “shield” charges on proteins so weak attractive force
high [salt]
ions compete for water allowing hydrophobic and charge interactions
ion exchange chromatography
positively or negatively charged functional groups attached to resin
cation exchanger(negatively charged resin)
binds cations
anion exchanger (positively charged resin)
binds anions
what factors affect ion exchange chromatography?
net charge on molecule (greater net charge, better protein binding), solution pH, ionic strength (small ions compete with proteins for binding to the column)
size exclusion chromatography
separates molecules based on size/shape; resin matrix is porous with pores in a defined size range
analytical technique for protein MW estimation
protein MW can be estimated from a plot of elution volumes vs log MW. protein standards are run to define dependence of elution volume on size, then unknown protein is compared to standards
affinity chromatography
proteins bind to resin via specific interactions with ligands covalently bound to the resin
Polyacrylamide Gel Electrophoresis (PAGE)
protein is denatured by and coated with sodium docecyl sulfate (SDS); 1 SDS molecule for every 2 AAs; each SDS molecule is negatively charged so that SDS charge overwhelms the protein charge & separation is dependent on mass
how can protein pI can be determined?
gel containing a pH gradient; proteins will be moved thru gel by electric field until they reach a region of pH where pI=pH; called ISOELECTRIC FOCUSING
isoelectric focusing and SDS gel electrophoresis can be combined to do what?
to separate proteins according to their mass and pI in this 2D method
