Flashcards in BIOCHEM 3 - Separation Techniques Deck (45):
Sources of Starting Material
a. Animal or plant tissues (ex. Myoglobin from beef muscle)
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.
– liberation of biomolecule to the cell
-process of breaking cells open to release organelles and obtain a pure sample
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
a type of mechanical disruption in which there's the use of ultrasonic vibrations
a type of mechanical disruption in which the cell wall is ripped from the cell when the material undergoes rapid vibration with glass beads
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
2. Osmotic lysis (for animal cells)
methods of solubilization
Osmotic lysis (for animal cells)
principle of isoelectric ppt
At ____ protein will agglomerate and you can now precipitate the protein
Solubility of a protein at low ionic strength generally increases with the salt concentration.
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.
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
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.
• When macromolecular solution is forced under pressure thru a semi-permeable bag/disc
• Column is packed with porous beads.
D. Gel Filtration Chromatography
• Small molecules enter the beads and are retarded, while, large molecules cannot enter and so they migrate faster.
D. Gel Filtration Chromatography
• With special disk and funnel
• 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:
Mobile phase- least polar
Stationary phase- polar
Normal Phase chromatography
Mobile phase- polar
Stationary phase- least polar
Reverse Phase chromatography
• A procedure based on the ability of proteins to interact with specific molecules
• It is the separation of charged particles in an electric field thru a support medium.
• Involves electrophoresis of protein mixtures thru stable ph gradient medium.
• Protein will migrate to the medium where ph = Iph
2. Isoelectric focusing
– rate of movement of particles is proportional to their net charge and inversely related to their size
3. Gel electrophoresis
types of Gel electrophoresis
• Agarose Gel Electrophoresis (AGE)
• Polyacrylamide Gel Electrophoresis (PAGE)
- mask the intrinsic charge of protein due to large negative charge it imparts on it
-a surfactant; breaks disulfide bridges
In SDS PAGE, which proteins will migrate easily
those with low MW
• Similar to affinity chromatography but it contains a resin
Column is packed with resin that have ligand (either positive or negative in charge)
type of chromatography wherein Interaction is based on net charge
Resin of cation exchanger chrom
first elution of cation exchanger chrom
last elution of cation exchanger chrom
Resin of anion exchanger chrom
first elution of anion exchanger chrom
last elution of anion exchanger chrom
AA is + charged
when ph = pI
AA is zwitterion