BIOCHEM 3 - Separation Techniques Flashcards Preview

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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

AA is + charged

44

when ph = pI

AA is zwitterion

45

when ph > pI

AA is - charged

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