Source: Stryer, figure 4.2

• separation based upon molecular size

Source: Stryer, figure 4.3

• column is filled with semi-solid beads of a polymeric gel that will admit ions and small molecules into their interior but not large ones

• when a mixture of molecules and ions dissolved in a solvent is applied to the top of the column, the smaller molecules (and ions) are distributed through a larger volume of solvent than is available to the large molecules

• consequently, the large molecules move more rapidly through the column, and in this way the mixture can be separated (fractionated) into its components

• the porosity of the gel can be adjusted to exclude all molecules above a certain size

• Sephadex, Sepharose or Sephacryl, which are fine porous beads, are trade names for gels that are available commercially in a broad range of porosities

• on the basis of their density and sedimentation velocity

• separation by filters with estimated pore size

Source: Stryer, figure 4.4

Source: Stryer, figure 4.4

• electrophoretic separation of proteins is most commonly performed in polyacrylamide gels
• separation of molecules on the polyacrylamide gel matrix by applying an electric field
  
  • polyacrylamide gels:
    • successful separation can be accomplished by electrophoresis in various gels (semisolid suspensions in water) rather than in a liquid solution
    • gels are cast between a pair of glass plates by polymerizing a solution of acrylamide monomers into polyacrylamide chains and simultaneously cross-linking the chains into a semisolid matrix
    • gel pore size can be varied by adjusting the concentrations of polyacrylamide and the cross-linking reagent
      • highly cross-linked polyacrylamide gel = pores are quite small
      • such a gel could resolve small proteins and peptides, but large proteins would not be able to move through it
    • smaller proteins migrate faster than larger proteins through the gel
    • gel's pore size and strength of the electric field influence the rate of movement

• sodium dodecyl sulfate (SDS) coats proteins with negative charges
• coated polypeptide chains can then separated by molecular mass (method to determine molecular weight)

• determine the approximate molecular weight of a polypeptide chain as well as the subunit composition of a complex protein
  • even chains that differ by less than 10 percent in molecular weight can be separated !
• molecular weight estimation by distance comparison
  • compare distance 1 (migration through the gel) with distance 2 (migration of proteins with known molecular weight)

  separation procedure:

  • proteins are exposed to the ionic detergent SDS before and during gel electrophoresis
  • SDS denatures proteins, causing multimeric proteins to dissociate into their subunits
  • all polypeptide chains are then forced into extended conformations with similar charge / mass ratios
  • SDS treatment eliminates the effect of differences in shape
    • chain length = unique determinant of the migration rate of proteins
  • individual polypeptide chains migrate as a negatively charged SDS-protein complex through the porous polyacrylamide gel
  • speed of migration is proportional to the size of the proteins
    • smaller polypeptides running faster than larger polypeptides

(see also chapter: protein denaturation - changes in salt concentration)