Flashcards in BIOCHEM 3 - Separation Techniques Deck (45):
1
Sources of Starting Material
a. Animal or plant tissues (ex. Myoglobin from beef muscle)
b. Microorganisms
2
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
3
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
4
– liberation of biomolecule to the cell
-process of breaking cells open to release organelles and obtain a pure sample
Homogenization
5
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
6
a type of mechanical disruption in which cells are forced through a small hole under very high pressure
french press
7
a type of mechanical disruption in which there's the use of ultrasonic vibrations
Ultrasound/sonication
8
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
9
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)
10
methods of solubilization
mechanical disruption
Osmotic lysis (for animal cells)
11
principle of isoelectric ppt
At ____ protein will agglomerate and you can now precipitate the protein
zwitterion,
12
Solubility of a protein at low ionic strength generally increases with the salt concentration.
Salting in
13
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–
14
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
15
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
16
for separation of crude mixtures of cellular components
Differential Centrifugation –
17
basis of Differential Centrifugation –
-basis: molecular size
18
• 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
19
• When macromolecular solution is forced under pressure thru a semi-permeable bag/disc
C. Ultrafiltration
20
• Column is packed with porous beads.
D. Gel Filtration Chromatography
21
• Small molecules enter the beads and are retarded, while, large molecules cannot enter and so they migrate faster.
D. Gel Filtration Chromatography
22
• With special disk and funnel
C. Ultrafiltration
23
• Separation of molecules in a mixture depends on the affinity to either mobile or stationary phase.
A. Chromatography
24
• Types of chromatography based on the polarity of each phase:
normal phase
reverse phase
25
Mobile phase- least polar
Stationary phase- polar
Normal Phase chromatography
26
Mobile phase- polar
Stationary phase- least polar
Reverse Phase chromatography
27
• A procedure based on the ability of proteins to interact with specific molecules
Affinity Chromatography
28
• It is the separation of charged particles in an electric field thru a support medium.
A. Electrophoresis
29
• Involves electrophoresis of protein mixtures thru stable ph gradient medium.
• Protein will migrate to the medium where ph = Iph
2. Isoelectric focusing
30
– rate of movement of particles is proportional to their net charge and inversely related to their size
3. Gel electrophoresis
31
types of Gel electrophoresis
• Agarose Gel Electrophoresis (AGE)
• Polyacrylamide Gel Electrophoresis (PAGE)
32
- mask the intrinsic charge of protein due to large negative charge it imparts on it
-a surfactant; breaks disulfide bridges
SDS
33
In SDS PAGE, which proteins will migrate easily
those with low MW
34
• Similar to affinity chromatography but it contains a resin
•
Ion exchanger
35
Column is packed with resin that have ligand (either positive or negative in charge)
Ion exchanger
36
type of chromatography wherein Interaction is based on net charge
Ion exchanger
37
Resin of cation exchanger chrom
anion resin
38
first elution of cation exchanger chrom
anion
39
last elution of cation exchanger chrom
cation
40
Resin of anion exchanger chrom
cation
41
first elution of anion exchanger chrom
cation
42
last elution of anion exchanger chrom
anion
43
when ph < pI
AA is + charged
44
when ph = pI
AA is zwitterion
45