BIOCHEM 3 - Separation Techniques

Question 1

Sources of Starting Material

Answer 1

a. Animal or plant tissues (ex. Myoglobin from beef muscle)

b. Microorganisms

Question 2

Criteria for choosing a sample

Answer 2

ease of obtaining sufficient quantity of tissue
amount of biomolecule in the tissue
any properties peculiar to the biomolecule of choice

Question 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.

Answer 3

Isoelectric precipitation

Question 4

– liberation of biomolecule to the cell

-process of breaking cells open to release organelles and obtain a pure sample

Answer 4

Homogenization

Question 5

types of Mechanical Disruption

Answer 5

a. French press –
b. Ultrasound/sonication –
c. Beadmill –
d. Use of high speed blender
e. Grinding with sand or alumina
f. Hand homogenizer

Question 6

a type of mechanical disruption in which cells are forced through a small hole under very high pressure

Answer 6

french press

Question 7

a type of mechanical disruption in which there’s the use of ultrasonic vibrations

Answer 7

Ultrasound/sonication

Question 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

Answer 8

Beadmill

Question 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

Answer 9

2. Osmotic lysis (for animal cells)

Question 10

methods of solubilization

Answer 10

mechanical disruption
Osmotic lysis (for animal cells)

Question 11

principle of isoelectric ppt

At ____ protein will agglomerate and you can now precipitate the protein

Answer 11

zwitterion,

Question 12

Solubility of a protein at low ionic strength generally increases with the salt concentration.

Answer 12

Salting in

Question 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

Answer 13

Isoelectric ph or point–

Question 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.

Answer 14

Salting out

Question 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

Answer 15

centrifugation

Question 16

for separation of crude mixtures of cellular components

Answer 16

Differential Centrifugation –

Question 17

basis of Differential Centrifugation –

Answer 17

-basis: molecular size

Question 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.

Answer 18

dialysis

Question 19

• When macromolecular solution is forced under pressure thru a semi-permeable bag/disc

Answer 19

C. Ultrafiltration

Question 20

• Column is packed with porous beads.

Answer 20

D. Gel Filtration Chromatography

Question 21

• Small molecules enter the beads and are retarded, while, large molecules cannot enter and so they migrate faster.

Answer 21

D. Gel Filtration Chromatography

Question 22

• With special disk and funnel

Answer 22

C. Ultrafiltration

Question 23

• Separation of molecules in a mixture depends on the affinity to either mobile or stationary phase.

Answer 23

A. Chromatography

Question 24

• Types of chromatography based on the polarity of each phase:

Answer 24

normal phase

reverse phase

Question 25

Mobile phase- least polar | Stationary phase- polar

Answer 25

Normal Phase chromatography

Question 26

Mobile phase- polar | Stationary phase- least polar

Answer 26

Reverse Phase chromatography

Question 27

• A procedure based on the ability of proteins to interact with specific molecules

Answer 27

Affinity Chromatography

Question 28

• It is the separation of charged particles in an electric field thru a support medium.

Answer 28

A. Electrophoresis

Question 29

* Involves electrophoresis of protein mixtures thru stable ph gradient medium. * Protein will migrate to the medium where ph = Iph

Answer 29

2. Isoelectric focusing

Question 30

– rate of movement of particles is proportional to their net charge and inversely related to their size

Answer 30

3. Gel electrophoresis

Question 31

types of Gel electrophoresis

Answer 31

* Agarose Gel Electrophoresis (AGE) | * Polyacrylamide Gel Electrophoresis (PAGE)

Question 32

- mask the intrinsic charge of protein due to large negative charge it imparts on it - a surfactant; breaks disulfide bridges

Answer 32

SDS

Question 33

In SDS PAGE, which proteins will migrate easily

Answer 33

those with low MW

Question 34

• Similar to affinity chromatography but it contains a resin | •

Answer 34

Ion exchanger

Question 35

Column is packed with resin that have ligand (either positive or negative in charge)

Answer 35

Ion exchanger

Question 36

type of chromatography wherein Interaction is based on net charge

Answer 36

Ion exchanger

Question 37

Resin of cation exchanger chrom

Answer 37

anion resin

Question 38

first elution of cation exchanger chrom

Answer 38

anion

Question 39

last elution of cation exchanger chrom

Answer 39

cation

Question 40

Resin of anion exchanger chrom

Answer 40

cation

Question 41

first elution of anion exchanger chrom

Answer 41

cation

Question 42

last elution of anion exchanger chrom

Answer 42

anion

Question 43

when ph < pI

Answer 43

AA is + charged

Question 44

when ph = pI

Answer 44

AA is zwitterion

Question 45

when ph > pI

Answer 45

AA is - charged