Professional Documents
Culture Documents
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.