BIOCHEM 3 - Separation Techniques Flashcards
Sources of Starting Material
a. Animal or plant tissues (ex. Myoglobin from beef muscle)
b. 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
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
– liberation of biomolecule to the cell
-process of breaking cells open to release organelles and obtain a pure sample
Homogenization
types of Mechanical Disruption
a. French press –
b. Ultrasound/sonication –
c. Beadmill –
d. Use of high speed blender
e. Grinding with sand or alumina
f. Hand homogenizer
a type of mechanical disruption in which cells are forced through a small hole under very high pressure
french press
a type of mechanical disruption in which there’s the use of ultrasonic vibrations
Ultrasound/sonication
a type of mechanical disruption in which the cell wall is ripped from the cell when the material undergoes rapid vibration with glass beads
Beadmill
method of solubilization
– involves suspension of cells in a hypotonic solution
- a hypotonic solution contains a semi-permeable membrane
- cells will swell and eventually burst
- organelles will come out of the cell, thus isolating the protein
- Osmotic lysis (for animal cells)
methods of solubilization
mechanical disruption Osmotic lysis (for animal cells)
principle of isoelectric ppt
At ____ protein will agglomerate and you can now precipitate the protein
zwitterion,
Solubility of a protein at low ionic strength generally increases with the salt concentration.
Salting in
ph at which a molecule carries no net charge
- point at which the protein has reduced solubility
- protein is unable to react with medium and so it will fall out of the solution
Isoelectric ph or point–
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.
Salting out
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
centrifugation
for separation of crude mixtures of cellular components
Differential Centrifugation –
basis of Differential Centrifugation –
-basis: molecular size
- A process that separates molecules by the use of semi-permeable membrane.
- It is the movement of molecules by diffusion from high concentration to low concentration.
dialysis
