Ch. 5 - Protein Purification & Characterization Techniques

Homogenization & techniques

breaking cells open to get organelles

• grind tissue in blender w/ suitable buffer

• use homogenizer

• sonication (sound waves)

differential centrifugation

when ruptured cells are centrifuged many times & ⬆ F_g every time to separate particles

salting out

purification technique based on differential solubility in salt soln

• NH4SO4 used—makes protein less soluble by ⬆ hydrophobic interactions

• depends on overall charge, ionic strength, & polarity

Chromatography

technique based on the fact that compounds distribute themselves between diff. phases

Stationary phase

substance that slows down flow of sample, facilitating separate

Mobile phase

portion of system in which the sample moves through (usually liquid)

Affinity Chromatography

column separation based on specific binding of molecules to ligand

• bead will only bind to specific desired protein (noncovalent)

• would elute last

Ion-Exchange Chromatography & the 2 ion-exchange resins

separating substances based on charge

• cation exchanger: resin that has – charge to bind to + molecules

• anion exchanger: resin that has + charge to bind to – molecules

Size-exclusion or Gel-filtration Chromatography

technique used to separate substances based on size

• cross-linked beads → small enough particles enter (elute slower), big molecules don’t enter (elute faster)

Electrophoresis

method for separating molecules based on charge:size

• agarose for nucleic acids & polyacrylamide for proteins

SDS-PAGE

electrophoresis technique where proteins are separated on size

• all proteins – bc SDS used to treat it