chromatography(l13 & l14)

Question 1

Assays should be

Answer 1

sensitive, specific, rapid, quantitative

Question 2

Activity

Answer 2

U of enzyme per ml

Question 3

Specific activity

Answer 3

U per mg of protein

Question 4

Yield

Answer 4

(activity after purification)/(activity before purification)

Question 5

purity

Answer 5

(S.A. after purification)/(specific after before purification)
(is a fold increase)

Question 6

As [Salt] increases

Answer 6

the + and - ions will compete with surface amino acids for water, (proteins aggregate(condense))
- (salting in can still increases solubility a little bit)

Question 7

dialysis tubing

Answer 7

has pores with a specific molecular weight cut off so salt can pass through cause it’s small
- excess salt can impede subsequent isolation steps

Question 8

mobile phase

Answer 8

the carrier for the compounds to be seperated

Question 9

stationary phase

Answer 9

a substance that the compounds to be seperated pass by or interact with

Question 10

packing

Answer 10

opening the stopper and letting some liquid run out to make the stationary phase more densely/evenly packed

Question 11

loading

Answer 11

the sample of interest is placed at the top of the stationary phase

Question 12

chromatography steps

Answer 12

pouring, packing, loading, running, collecting

Question 13

What proteins do not interact with the beads

Answer 13

the smallest

Question 14

partition coefficient

Answer 14

standardizes elution measurements (Ve-Vo)/(Vt-Vo)

Question 15

relative molecular weight

Answer 15

refers to the shape of the protein

Question 16

Samples become diluted from

Answer 16

turbulence, diffusion, friction

Question 17

When column is negative it contains

Answer 17

cation exchangers

Question 18

stepwise gradient elution

Answer 18

alter the pH to reduce the charge of the proteins bound to the column
increase the salt concentration to increase competition for binding the stationary phase

Question 19

Electrophoresis

Answer 19

migration of ions in an electric field

Question 20

electrophoresis proportionality

Answer 20

directly proportional to charge, inversely proportional to size and shape

Question 21

purposes of the gel in gel electrophoresis

Answer 21

slows down movement, prevents diffusion of proteins, creates seperation

Question 22

SDS

Answer 22

a detergent that will denature proteins and give them a negative charge

Question 23

acrylamide

Answer 23

a plastic monomer, used industrially, that can be chained together

Question 24

Acrylamide gel ingredients

Answer 24

acrylamide(to form chains)
bisacrylamide(branch chains/crosslinking)
persulfate(for buffered salt solution, free radicals to catalyze polymerization)
temed(for buffered salt solution, free radicals to catalyze polymerization)

Question 25

Gel stains

Answer 25

Coomasie blue staining(10 mins, soak in buffer for 10 mins)(x 2 or 3)
silver nitrate staining(more sensitive, complicated and time consuming, 11 solution changes, 2+ hours)
fluorescent dye staining(sensitive, requires special equipment)