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I. Protein Isolation
Selection of Starting Material
Sources
a. animal or plant tissues
b. microorganisms
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
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.
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.
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.