Class 7 E1

Question 1

Why use protein purification?

Answer 1

research, use in drugs, additives in detergents, foods, industrial processes.

Question 2

As purification proceeds then?

Answer 2

of components / contaminants decreases.

Question 3

Protein purification depends on?

Answer 3

size / charge.
location in cell (nucleus, membrane, mitochondrion)
solubility (hydrophobic / philic).

Question 4

When do you test the purity of the same?

Answer 4

at the end.

Question 5

Steps of protein purification?

Answer 5

1. homogenize cells.
2. crude separation.
   3, 4, 5. refined separation + purification steps.
3. access success.

Question 6

protein purification: fold purification

Answer 6

amount of protein of interest in final sample / amount in crude sample.

Question 7

protein purification: specific activity

Answer 7

measure of enzyme purity, units catalyzed / time / mg enzyme.

Question 8

protein purification: homogenization (sonification)

Answer 8

grinding / disrupting tissue to make uniform sample.

Question 9

protein purification: centrifugation

Answer 9

spinning tube fast enough so heavier particles separate from lighter particles.

Question 10

protein purification: yield (%)

Answer 10

total amount of protein (or activity) in final sample / amount of protein (or activity) in crude sample.

Question 11

salting in / out

Answer 11

basic (not refined) way to separate class A from B.

Question 12

Salt amount in salting in / out?

Answer 12

little - helps keep charged molecules in soln.

a lot - makes charged molecules precipitate.

Question 13

What are the 3 methods used to purify proteins?

Answer 13

1. Salting out
2. Column-based methods
3. Immunoprecipitation

Question 14

What are the 3 types of column-based methods?

Answer 14

1. size-exclusion chromatography
2. ion-exchange chromatography
3. affinity chromatography

Question 15

chromatography

Answer 15

movement of a compound w/ mobile phase and stationary phase.

Question 16

chromatography: mobile phase

Answer 16

medium, compound dissolved and applied to stationary phase (buffer).

Question 17

chromatography: stationary phase

Answer 17

immobile / matrix over which mobile phase containing compound flows (“matrix””

Question 18

Separation occurs in chromatography bc?

Answer 18

diff compounds have diff relative affinities for mobile vs. stationary phases.

Question 19

chromatography columns

Answer 19

allow separation of protein soln into fractions.

Question 20

chromatographic media

Answer 20

resins / beads, microscopic - form support / separation chemistry, size of beads (mesh size) = diameter of any particle - #’s reciprocally related.

Question 21

What are placed in chromatography columns?

Answer 21

resins

Question 22

size exclusion chromatography

Answer 22

separates proteins based on size

Question 23

The gel beads in size exclusion chromatography have pores of a defined size range that?

Answer 23

allow smaller molecules to enter while excluding larger molecules.

Question 24

Which proteins reside longer in the gel beads in size exclusion chromatography?

Answer 24

smaller ones.

Question 25

Which proteins elute in early fractions of size exclusion chromatography?

Answer 25

large ones.

Question 26

ion exchange chromatograpahy

Answer 26

separates based on charge

Question 27

the stationary phase of ion exchange chromatography is?

Answer 27

charged and binds proteins of opposing charge.

Question 28

In ion exchange chromatography you collect?

Answer 28

fractions of eluate from column over time.

Question 29

In ion exchange chromatography you can elute using what?

Answer 29

salt, pH change.

Question 30

What are the steps of ion exchange chromatography?

Answer 30

1. add mix of AA's or proteins into an immobilized cationic (+) / anionic (-) stationary phase. 2. the opp charged proteins will bind to the charged matrix as they flow through. 3. Elute using a specific buffer.

Question 31

affinity chromatography

Answer 31

isolates specific proteins and purifies fusion proteins.

Question 32

Affinity chromatography takes advantage of?

Answer 32

specific interactions btw protein and stationary phase.

Question 33

The stationary phase in affinity chromatography can be loaded with?

Answer 33

a ligand or antibody; natural ligand of protein / antibody.

Question 34

Some of the epitope tags that can be engineered to express the vector or transgene in affinity chromatography.

Answer 34

1. 6x-His 2. GST 3. MBP 4. Protein A etc.

Question 35

Steps of using affinity chromatography to purify fusion proteins.

Answer 35

1. amylose resin matrix put into column. 2. run homogenate over column. 3. wash of other proteins. 4. collect fractions

Question 36

affinity chromatography: Factor X

Answer 36

can cleave and release pure target protein.

Question 37

immunoprecipitation

Answer 37

uses antibodies

Question 38

Immunoprecipitation uses?

Answer 38

Ig Fc linked to beads

Question 39

The Ig Fc linked beads in immunoprecipitation work like?

Answer 39

the stationary phase in a column similar to how Ig's are embedded in the B cell membrane.

Question 40

The Ig Fc linked beads are heavy bc?

Answer 40

they allow quick separation via quick centrifugation spin (<1 min)

Question 41

Steps to immunoprecipitation?

Answer 41

1. prepare sample. 2. capture antibody. 3. wash and elute.