Molecular separations

Question 1

reasons for biomolecule purification

Answer 1

study their properties
analyse distribution/ abundance
commercial/ medical use

Question 2

synthetic biomolecules

Answer 2

incomplete reaction products resembling final product

Question 3

2 main techniques for purification

Answer 3

electrophoresis
chromatography

Question 4

relative centrifuge force

Answer 4

centrifugal force/ g force

Question 5

microfuge

Answer 5

low speed

Question 6

ultracentrifuge

Answer 6

high speed

Question 7

isolation of protein sources in blood/ bacteria

Answer 7

centrifuge

Question 8

isolation of protein sources in organs/ tissues

Answer 8

homogenisation

Question 9

cell g force

Answer 9

3000g

Question 10

organelle g force

Answer 10

20000g

Question 11

protein g force

Answer 11

100000g

Question 12

chromatography factors

Answer 12

size
charge
affinity

Question 13

factors affecting affinity for stationary phase

Answer 13

charge interactions (ion exchange chromatography)
hydrophobicity (reverse phase/ hydrophobic interaction)
size (gel filtration/ size exclusion)
specific binding (affinity chromatography)

Question 14

gel filtration

Answer 14

porous beads in which size affects distance travelled

Question 15

ion exchange chromatography

Answer 15

elution via salt conc ^ competing for charge interactions
protein rich in charged residues

Question 16

reverse phase chromatography

Answer 16

eluted by polarity reversal of solution via solvents

Question 17

affinity chromatography

Answer 17

addition of free binding partner

Question 18

immobilised metal affinity chromatography

Answer 18

1. Histag addition
2. elution
   *imidazole addition

Question 19

histag

Answer 19

6-10 histidine residues at N/C terminus end
imidazole side chain specifically binding to metals

Question 20

addition of histag

Answer 20

addition of DNA coding for histag into natural DNA
bacteria produce protein

Question 21

histidine characteristic

Answer 21

heteroaromatic
- charged amine group

Question 22

histag elution

Answer 22

imidazole addition elutes
gradient/ step addition

Question 23

step elution

Answer 23

all bound molecules elute at once

Question 24

gradient elution

Answer 24

separates if different types bound

Question 25

gel electrophoresis gel function

Answer 25

convection current prevention so molecules move as a band, not dispersed

Question 26

gel pore size effect on gel electrophoresis

Answer 26

control macromolecule migration speed

Question 27

PAGE (polyacrylamide gel electrophoresis)

Answer 27

acrylamide polymers cross-linked w methylene bisacrylamide

Question 28

acrylamide polymer percentage effect

Answer 28

changes pore size

Question 29

pH of PAGE

Answer 29

>8 therefore proteins are mostly negatively charged

Question 30

NATIVE-PAGE

Answer 30

Proteins separated by size, shape and charge

Question 31

colourless protein reveal in NATIVE-PAGE

Answer 31

blue-stain

Question 32

SDS

Answer 32

sodium dodecyl sulphate *lauryl sulphate in shampoo

Question 33

SDS process

Answer 33

denatures proteins/ adds negative charge shape/charge independent heated w SDS/ mercapto-ethanol to disrupt disulfide bonds and denature

Question 34

SDS-PAGE

Answer 34

separation by protein size only mercapto ethanol and heated to 95 degrees celcius denature depends on pore size marker proteins often used

Question 35

pore size proportion to molecular weight in SDS-PAGE

Answer 35

pore size proportional to log(MW)

Question 36

IEF

Answer 36

Iso-Electric Focussing

Question 37

IEF summary

Answer 37

Native-page carried out on pH gradient caused by ampholytes

Question 38

ampholytes

Answer 38

large polyions

Question 39

equilibrium technique IEF

Answer 39

proteins stop when they reach an isoelectric point

Question 40

what does IEF identify

Answer 40

proteins, their isomers and chemical modification

Question 41

2D Gel electrophoresis

Answer 41

IEF in tubular gels as first dimension, SDS page in second dimension

Question 42

isoelectric point/ pI

Answer 42

pH at which a particular molecule carries no net electrical charge

Question 43

what does 2D gel electrophoresis separate by?

Answer 43

size and pI

Question 44

DNA gel electrophoresis visualisation

Answer 44

UV light and fluorescent dyes

Question 45

DNA gel electrophoresis

Answer 45

small DNA fragments separated at high res via acrylamide gels in sequencing larger fragements have larger pore gels fixed negative charge density