Lecture 19 - Protein purification

Question 1

For enzymes (protein catalysts), the assay usually measures

Answer 1

enzyme activity

Question 2

Salt concentration is expressed as

Answer 2

ionic strength

Question 3

At low ionic strength, protein solubility generally

Answer 3

increases with the salt concentration (salting in)

Question 4

At high ionic strength, protein solubility generally

Answer 4

decreases with the salt concentration (salting out)

Question 5

Proteins are generally the least soluble at the

Answer 5

pI (isoelectric point)

Question 6

Uncharged proteins are _______ to the salt concentrations

Answer 6

insensitive

Question 7

Water-miscible solvents such as acetone and ethanol are good

Answer 7

protein precipitants

Question 8

The first experimental step in protein purification is to

Answer 8

get it out of the cell and into solution (cell lysis)

Question 9

Most of the procedures for lysing cells use some variation of___________ followed by ________

Answer 9

crushing or grinding; filtration and/or centrifugation to remove large, insoluble particles

Question 10

After homogenization, the tissue is fractionated by

Answer 10

several centrifugation steps

Question 11

After centrifugation, the next step is often

Answer 11

salting out of the proteins

Question 12

Proteins are generally less soluble at _______ salt concentratinos

Answer 12

higher

Question 13

Salting out is usually done with

Answer 13

ammonium sulfate

Question 14

After salting out, salt can be removed by

Answer 14

dialysis

Question 15

After salting out and dialysis, ________ is usually performed

Answer 15

chromatography

Question 16

Gel filtration separates molecules based on

Answer 16

size

Question 17

In gel filtration chromatography, large molecules travel

Answer 17

faster

Question 18

Ion-exchange chromatography separates molecules based on

Answer 18

charge

Question 19

In ion-exchange chromatography, bound proteins are eluted by

Answer 19

increasing the concentration of ions that compete with the protein for binding to the column

Question 20

Hydrophobic interaction chromatography purifies

Answer 20

nonpolar proteins

Question 21

Affinity chromatography separates molecules based on

Answer 21

their affinity for the column material

Question 22

In metal chelate affinity chromatography,

Answer 22

a divalent metal ion (2+) is attached to the matrix

Question 23

In affinity chromatography, the protein can be eluted by

Answer 23

imidazole