 Western blotting:

o Electrophoresis
o Transfer onto polymer sheet
o Primary antibody
o Secondary antibody with fluorescent tag
 ELISA: use of antibodies to quantify the amount of proteins in a sample, enzyme that
reacts with colourless substrate to produce a colour, the presence of colour indicates
the presence of the antigen.
o Polyclonal: antibodies are derived from multiple antibody producing cell
populations, mixture of antibodies each specific to one of the epitopes on an
antigen
o monoclonal: antibodies are identical produced by clones of the same antibody,
they recognize one specific epitope
 Green fluorescent protein: cells can be stained with fluorescent antibody which reveals
location of protein and hints at function
 X-ray chromatography: clearest visualization of precise 3D position of most atoms within
a protein,
o Protein/protein complex forms high order crystals
o Narrow X-ray beam is directed at crystals some scattered
 Amplitude of scattered waves=# of electrons
 Each diffracted contains scattered waves if waves reinforce one another,
they are in phase if not they are out of phase
 The way the scattered waves recombine = atomic arrangement
o Allows to produce electron density map to determine the position of atoms in
crystalized molecule
 1D NMR: post powerful method for determining protein structures, can revel atomic
structure of macromolecule in a solution as long as its highly concentrated,
 2D NMR: how spins of protons affect their neighbor by altering the spin of one nucleus
using radio frequency. Graphically displays pairs of protons in close proximity.
 Cryo-electron microscopy:
o thin layer of protein solution prepped and frozen.
o Sample exposed to incident electron beam in vacuum, each protein interacts
with the beam to produce 2D projection, many detected each projecting
differently
o Computer merges projections to form 3D model
o Allows for identification of binding sites and visualize of large molecular
complexes
 Thermodynamics:
o Reaction is spontaneous when G is negative
o Equilibrium when G is zero
o Not spontaneous when G is positive
o G+ determines rate, enzymes lower G+ but not G
o S is the change in entropy, -S= not spontaneous +S= spontaneous
 Kcat is catalytic, Kcat/Km is a measure of catalytic energy
 Competitive: Vmax same, Km increase
 Uncompetitive: Vmax decrease, Kmax same
 non-competitive: Vmax decrease and Km decrease