User:Benjah-bmm27/degree/3/CA

=The Analysis of Macromolecules=

Separation of macromolecules

 * Size exclusion chromatography (SEC)
 * AKA gel filtration chromatography or gel permeation chromatography (GPC)
 * Stationary phase is a porous material called Sephadex
 * Sephadex is cross-linked dextran, a polymer of glucose
 * Smaller molecules can penetrate every pore of the stationary phase, so are retained longer
 * Larger molecules cannot penetrate any of the pores, so elute faster
 * YouTube animation of SEC in action: http://www.youtube.com/watch?v=wjuWb6XpRmQ
 * It is possible to calculate the approximate molecular weight of a macromolecule using SEC
 * Determine the column void volume, Vo (volume a particle sees if it cannot penetrate any pores) by flowing dextran blue (a huge molecule, m.w. 2 MDa) through the column
 * Determine elution volumes Ve of several macromolecules of known molecular weight
 * Plot a calibration curve: log(m.w.) vs. Ve/Vo
 * Measure Ve of the analyte (a macromolecule of unknown m.w.), read off m.w. from calibration curve
 * Stokes radius

FRET

 * Förster resonance energy transfer

SEM

 * Scanning electron microscopy (SEM)
 * Fire high-energy electrons at a sample in a vacuum chamber
 * Electron beam rasters across the sample
 * Detect backscattered electrons and secondary electrons
 * Backscattering is greater for atoms of higher atomic number

TEM

 * Transmission electron microscopy (TEM)