Summary of protein sequencing techniques
Primary Structure Determination
Method
Separation basis
Separation
Chromatography
Size-exclusion (Gel
Filtration)
-porous beads
Stokes radius
Stokes radius - radius of the
sphere they
occupy as
they tumble
in solution
proteins with larger SR elutes out first
Column
Chromatography
High-pressure
Liquid
Chromatography
(HPLC)
Stationary phase
– matrix of small
beads
Mobile phase
– percolates
through a
column uses
gravity (drip)
matrix is forced
through high
pressures
Ion-exchange
Charge-charge interaction
Proteins with weaker interactions elutes
out first
Hydrophobic Interaction
Tendency to associate with a
stationary phase covered with
hydrophobic groups
Proteins with more hydrophilic
character elutes out first
Affinity
High selectivity of proteins with
their ligands
Only the proteins that interact with the
immobilized ligands adhere
Selective precipitation
– exploits differences in relative
solubility of individual proteins as a
function of pH (isoelectric
precipitation), polarity
(precipitation with ethanol or
acetone), or salt concentration
(salting out with ammonium
sulfate)
Polyacrylamide Gel
Electrophoresis (PAGE)
-most widely used
1) Denaturation - Sodium Dodecyl
Sulfate (SDS) binds to proteins (1
molecule SDS per 2 peptide
bonds)
2) Electrophoresis separates
charged biomolecules based on
rates at which they migrate on
the electrical field
Large complexes – slower rate
(Higher molecular mass, lower rate)
Polypeptides in gel stained using
Coomassie blue and compared to a
standard of known molecular masses
Isoelectric Focusing (IEF)
Differences in isoelectric points
(pI)
Ampholytes – generates pH
gradient within a PA matrix
Proteins separate based on their pI in
the polyacrylamide gel pH gradient
