Protein Separation Methods- Ch. 4

column chromatography

separates protein mixture over a solid phase (porous matrix) using a liquid phase for mobility

column chromatography solid phase

lower affinity for solid phase: washes off first

higher affinity for solid phase: stays on column longer, washes off later

ion-exchange chromatography

proteins move through the charged column at different rates based on the sign and magnitude of net electric charge (opposing charges between column and protein move slower)

overall charge is dependent on primary structure and pH of solution

gel filtration chromatography

separates proteins based on size and shape → larger sized proteins take less time to pass through column beads

gel filtration pros and cons

proteins are left intact with good resolution, however it will not produce a pure solution and the flow rate is relatively slow

elution volume

each protein will have a different available volume for diffusion based on side and shape

elution volume protein sizes

larger protein: less volume needed

smaller protein: more volume needed

gel filtration pores

larger proteins will be excluded from pores and smaller proteins may enter the pores

going inside the pores gives the smaller proteins a longer distance to travel, therefore they will come out last

affinity chromatography

separates proteins based on their affinity/function

eluded by high concentrations of salt or ligand wash

leaves proteins intact

you must know the biological properties of the protein and ligand

the protein of interested is washed through the column last with ligand attached from ligand solution wash

best purification method

purification of proteins

except for affinity chromatography, no single method can purify a protein from a crude mixture. therefore, often several techniques ae employed to accomplish purification

hygroscopic salts

induces hydrophobicity → “salvation shell” reduces the ability of H-bonding in H2O

they sequester water from the protein and forces it to become less soluble

hydrophobic chromatography

separates proteins based on hydrophobicity, high concentrations of hygroscopic salts forces proteins to become less soluble and precipitate out

it also induces some soluble molecules into a non polar phase

the more hydrophobic the molecule, the less salt needed to promote binding → non polar molecules are extracted first

salting-in

at low concentrations, salt stabilized various charged groups on a protein molecule, enhancing solubility

salting-out

at high concentrations there is less water available to solubilize a protein, so the protein will precipitate out

final specific activity

starting specific activity ratio = purification factor

percent yield

percentage of final activity / percentage of starting activity

procedures from best to worst yield

1. crude cellular extract

2. precipitation with ammonium sulfate

3. ion-exchange chromatography

4. size-exclusion chromatography

5. affinity chromatography

electrophoresis

separation on an analytical scale → the proteins are pulled according to change.

the gel matrix hinders mobility based on size and shape

polyacrylamide

gel commonly used in electrophoresis

SDS

sodium dodecyl sulfate → a detergent that denatures proteins, giving them a uniformly negative charge

SDS PAGE

sodium-dodecyl-sulfate polyacrylamide gel electrophoresis, separates proteins only be molecular weight

the native shape and charge of proteins does not matter since they are denatured by SDS, the rate of movement depends only on size (smaller moves faster)

estimating molecular weight of a protein

can be determined by SDS page

plot of log(Mr) of marker proteins vs relative migration during electrophoresis = linear

two dimensional electrophoresis

permits resolution of complex protein mixtures

more sensitive than individual methods

isoelectric focusing to determine pI

a protein sample is applied to a gel with a pH gradient or a solution of ampholytes with the protein may be used to rehydrate a dehydrated gel

after staining, proteins are distributed along the pH gradient according to pI