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Separation and Characterization Techniques for Proteins and Amino Acids

I.

Protein Isolation
Selection of Starting Material
Sources
a. animal or plant tissues
b. microorganisms
Criteria for choosing a sample
a. ease of obtaining sufficient quantity of tissue
b. amount of biomolecule in the tissue
c. any properties peculiar to the biomolecule of choice
Methods of Solubilization
Homogenization liberation of biomolecule to the cell
A. Methods
1. Mechanical Disruption
a. French press cells are forced through a small hole under very high pressure
b. Ultrasound/sonication use of ultrasonic vibrations
c. Beadmill cell wall is ripped from the cell when the material undergoes rapid vibration with
glass beads
d. Use of high speed blender
e. Grinding with sand or alumina
f. Hand homogenizer
2. Osmotic lysis (for animal cells) involves suspension of cells in a hypotonic solution

II.

Purification of Proteins
Separation will be based on the characteristics of proteins.
1. Solubility
2. Molecular size, weight, density
3. Affinity
4. Charge
Methods of Separation and Purification
1. Based on Solubility
A. 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.
B. 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.

2. Based on Molecular Size, Weight and Density


A. 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.
Differential Centrifugation
For separation of crude mixtures of cellular components
B. Dialysis
A process that separates molecules by the use of a semi-permeable membrane.
It is the movement of molecules by diffusion from high concentration to low concentration.
C. Ultrafiltration

When macromolecular solution is forced under pressure thru a semi-permeable bag/disc.


D. 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
3. Based on Affinity
A. 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
B. Affinity Chromatography
A procedure based on the ability of proteins to interact with specific molecules.
4. Based on Charge
A. Electrophoresis

It is the separation of charged particles in an electric field thru a support medium.


1. Paper electrophoresis
2. Isoelectric focusing
Involves electrophoresis of protein mixtures thru stable pH gradient medium.
Protein will migrate to the region where pH = IpH.
3. Gel Electrophoresis rate of movement of particles is proportional to their net charge and
inversely related to their size
Agarose Gel Electrophoresis (AGE)
Polyacrylamide Gel Electrophoresis (PAGE)
SDS-PAGE
o SDS: mask the intrinsic charge of protein due to large negative charge it imparts on it.
o Separates protein in the order of their MWs.
B. 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)
Two types:
1. Anion Exchanger Chromatography
2. Cation Exchanger Chromatography