Separation and

Characterization
Techniques for Proteins
and Amino Acids
I. Protein Isolation
Selection of Starting Material
• Sources
– animal or plant tissues
– microorganisms
• Criteria for choosing a sample
– ease of obtaining sufficient quantity of
tissue
– amount of biomolecule in the tissue
– any properties peculiar to the
biomolecule of choice
Methods of Solubilization
Separation Techniques

Separation will be based on the

characteristics of biomolecules.

• Solubility
• Molecular size, weight, density
• Affinity
• Charge
Based on Solubility
• Change in pH
Isoelectric precipitation
A procedure in which the pH of the protein
mixture is adjusted to the pI of the protein
to be isolated to selectively minimize its
solubility.
Isoelectric
precipitation
• Change in ionic strength
Salting in
Solubility of a protein at low ionic
strength generally increases with
the salt concentration.
Salting out
Decrease in solubility of proteins and other
substances in aqueous solution at high ionic
strength. It is a result of the competition
between added salt ions and other dissolved
solutes for molecular solvation.
Based on Molecular Size
Centrifugation
Process of subjecting a suspension of
sample at greatly increased
gravitational field (centrifugal force) by
rapidly rotating a receptacle
containing the sample which will lead
to sedimentation of particles.
Application:
Differential Centrifugation
For separation of crude mixtures of
cellular components
Dialysis

• It is the movement of
molecules by diffusion
from high concentration
to low concentration.

• A process that separates molecules by the

use of a semi-permeable membrane.
Ultrafiltration
• When macromolecular solution is
forced under pressure thru a semi-
permeable bag/disc.
Gel Filtration Chromatography
• Column is packed
with porous beads
• Small molecules
enter the beads
and are retarded,
while, large
molecules cannot
enter and so they
migrate faster
Based on Affinity
Chromatography
• Separation of molecules in a
mixture depends on the affinity to
either mobile or stationary phase.
• Types of Chromatography based
on the polarity of each phase:
• Normal phase chromatography
• Reverse phase chromatography
Affinity Chromatography
A procedure based on the
ability of proteins to
interact with specific
molecules.
Based on Charge
• Electrophoresis
– It is the separation of charged
particles in an electric field thru a
support medium.
Isoelectric focusing
• Involves
electrophoresis of
protein mixtures
thru stable pH
gradient medium.
• Protein will
migrate to the
region where pH
= IpH.
Gel Electrophoresis

Types of Gel:
1. Agarose
2. Polyacrylamid
e
SDS-PAGE

• SDS: mask the

intrinsic charge of
protein due to
large negative
charge it imparts
on it.
• Separates protein
in the order of
their MWs.
Ion Exchanger Chromatography

• Similar to affinity
chromatography
• Interaction is based
on net charge
• Column is packed
with resin that
have ligand (either
positive or negative
in charge)
 Anion
Exchanger

Cation
Exchanger
Determination of 10 Structure of
Protein
A. Qualitative and Quantitative Analysis of Amino Acids
1. Hydrolyze peptide with 6N HCl at 110 C for 24 hours
2. Separate mixture by an amino acid analyzer
B. Determination of Amino Acid
Sequence
1.Methods for identification of N-
terminal amino acid residue
a. Sanger’s Reagent (DNFB)
b. Dabsyl chloride and Dansyl
chloride
c. Edman Degradation
• Sequentially
removes one residue
at a time from the
amino end.
• Phenyl
isothiocyanate reacts
with amino group to
form a
phenylthiocarbamoyl
derivative.
• Mild acid conditions
create cyclic
derivative
• Cyclic derivative is
separated by
chromatography to
identify amino acid
 d. Aminopeptidase

Gly – Arg – Phe – Ile – Lys – Met – Leu

2. Methods of Identification of C-
terminal amino acid residue
a. Carboxypeptidase

Gly – Arg – Phe – Ile – Lys – Met – Leu

b. Hydrazinolysis
3. Cleavage of Protein into Peptides
a. Chemical Method
Cyanogen bromide – cleaves at
carboxyl side of Met
b. Enzymatic Method
Trypsin - cleaves on carboxyl side of
Arg and Lys, but not when Pro is
present
Chymotrypsin - cleaves on carboxyl
side of aromatic amino acid, but not
when Pro is present