molecular separations

Question 1

why do we purify biomolecules

Answer 1

study properties
use medically
if they come from synthetic sources they contain incomplete reaction products that only resemble the final product

Question 2

centrifugation

Answer 2

larger the object, the quicker it sediments
easy to prepare cells from liquid
to prepare organelles, higher speed is needed
ultracentrifuge needed to prepare proteins

Question 3

mobile phase

Answer 3

liquid

Question 4

stationary phase

Answer 4

variable, differs depending on the type of chromatography being used

Question 5

why is the affinity for the stationary phase present?

Answer 5

charge interactions
hydrophobicity
size
specific binding (affinity)

Question 6

eluting

eluate

Answer 6

when molecules leave the column they are eluting

the sample is called the eluate

Question 7

separation by size

Answer 7

size exclusion chromatography

gel filtration chromatography

Question 8

separation by charge interactions

Answer 8

ion exchange chromatography

elution by increasing salt concentration which competes for the charged interaction

Question 9

separation by hydrophobicity

Answer 9

reverse phase chromatography
proteins with hydrophobic surface separated
eluted by reversing the polarity of the solution from polar to non polar with solvents such as ethanol.
molecules stick in water and are washed off in ethanol

Question 10

separation by specific binding

Answer 10

affinity chromatography

free binding partner added which elutes the proteins

Question 11

gel electrophoresis

Answer 11

gel used to prevent connection currents from dispersing molecules. molecules move as a band
gels pore size controls migration speed of macromolecules

Question 12

native PAGE

Answer 12

polyacrylamide gel eectrophoresis
uses polymers of acrylamide
gradient gels possible 
uses pH above 8 so most proteins are negatively charged 
= Native PAGE
PROTEINS SEPARATED BY INTRINSIC CHARGE

Question 13

SDS

Answer 13

found in shampoos as lauryl sulphate
denatures proteins
adds negative charge

Question 14

SDS-PAGE

Answer 14

separates proteins by size only
proteins adopt rod like shape
heating at 95 degrees ensures denaturation
DENATURED PROTEINS IN SDS SEPARATE ACCORDING TO SIZE

Question 15

IEF

Answer 15

iso electric focussing
native PAGE carried out on a pH gradient
proteins stop when they release isoelectric point
identifies proteins, their isomers and chemical modification

Question 16

2D gel electrophoresis

Answer 16

IEF in tubular gels as first dimension
SDS page as second
combines native page and sds page
CAN SEPARATE 100S OF PROTEINS IN ONE SAMPLE