protein purification

centrifugation

• 2 types of particles in suspension with different masses/densities will settle at the bottom of a test tube at different rates

• heavier molecules will settle more quickly than light ones

• sedimentation constant of a protein - measure of its sedimentation rate

differential centrifugation

• separates water-soluble proteins from insoluble cellular material

• forces cells to collect in pellet at the bottom while proteins remain in supernatant

• supernatant poured off and subjected to other purification techniques to separate other proteins it contains

rate-zonal centrifugation

• different water-soluble proteins separated according to their masses

• separated through density gradient - often created by conc sucrose solution

• proteins separate into different bands (zones) of masses as they travel at different rates through the gradient

gel electrophoresis

• separates molecules in a mixture under influence of an applied electric field

• dissolved molecules move at a speed determined by their mass to charge ratio

• smaller = move faster across gel, longer = move more slowly across gel

• longer, asymmetric structure = run more slowly across gel

• proteins within gel stained with organic/silver-based stain so that the position of protein can be identified

2D gel electrophoresis

• used to separate proteins of similar masses

• electric charge examined - determined by pH and in turn pKa

• proteins are separated by charge using isoelectric focusing and then separated by mass using SDS

• IEF - separate proteins according to their isoelectric points

liquid chromatography

• sample placed on top of tightly packed column of spherical beads held within a glass/metal/plastic cylinder

• sample flows down column and fractions collected which can be analysed for their contents and chemical activities

gel filtration chromatography

• samples separated by mass

• proteins flow around beads but smaller proteins can penetrate depressions more easily so pass out of column more slowly

• larger proteins can go around beads so pass out of column more quickly

• proteins of known mass used to produce calibration curve

ion-exchange chromatography

• used to separate proteins by charge

• specially modified beads covered by amino or carboxyl groups so that they have positive/negative charge

• buffer solution added to give proteins different charge depending on their isoelectric points

• greater attraction to beads = takes longer to pass through column

affinity chromatography

• separate proteins based on their ability to bind to other molecules

• affinity reagents such as ligands covalently attached to beads in the column

• column will only retain proteins that bind to molecule attached to beads

• proteins bound detached by exposure to detergents or changing salt concentration/pH so binding molecule disrupted

• can covalently modify proteins so that they bind to affinity reagent on beads

• histidine tag often used as affinity reagent- target protein binds to histidine tag, histidine tag small so does not interfere with protein function

SDS-PAGE gel electrophoresis

• gel which denatures proteins by binding to hydrophobic side chains

• makes protein linear and gives it negative charge - eliminates effect of differences in native conformation so chain length principal determinant in migration rate of proteins

• amount of SDS that binds to protein proportional to the length of the polypeptide chain