chapter 5 section 2

Question 1

On what basis can proteins be separated and purified?

Answer 1

By size and electrical charge

Question 2

When are proteins least soluble?

Answer 2

At their isoelectric point.

Question 3

What is “salting-in”?

Answer 3

Increasing ionic strength initially increases protein solubility.

Question 4

What is “salting-out”?

Answer 4

At higher salt concentrations, protein solubility decreases.

Question 5

How can protein concentration be estimated?

Answer 5

Using methods that reduce Cu²⁺ to Cu⁺, like the BCA assay.

Question 6

What does “specific activity” of an enzyme mean?

Answer 6

Enzyme activity per mg of protein.

Question 7

What is dialysis used for?

Answer 7

to separate small diffusible solutes from macromolecules using a semipermeable membrane.

Question 8

How does a dialysis bag work?

Answer 8

Solutes inside equilibrate with the bathing solution across the membrane.

Question 9

What principle does ion exchange chromatography use?

Answer 9

Separation based on charge.

Question 10

What separates molecules in gel filtration chromatography?

Answer 10

their size

Question 11

Which molecules elute first in gel filtration?

Answer 11

Larger molecules.

Question 12

What does SDS do in SDS-PAGE?

Answer 12

It denatures proteins and gives them a uniform negative charge.

Question 13

What does SDS-PAGE separate proteins by?

Answer 13

Molecular weight.

Question 14

How is protein mobility related to size in SDS-PAGE?

Answer 14

Smaller proteins move faster.

Question 15

What is two-dimensional gel electrophoresis

Answer 15

Separating proteins first by charge, then by size.

Question 16

How are proteins first separated in 2D electrophoresis?

Answer 16

By isoelectric focusing (charge).

Question 17

How are proteins separated in the second dimension of 2D electrophoresis?

Answer 17

By SDS-PAGE (size).

Question 18

Why use multiple purification steps?

Answer 18

To increase the specific activity and purity of a protein.

Question 19

What affects protein solubility besides pH?

Answer 19

Ionic strength and salt concentration.

Question 20

What is the main goal of protein purification?

Answer 20

To isolate a protein in a pure, functional form for study

Question 21

Why are proteins least soluble at their isoelectric point?

Answer 21

Because their net charge is zero, reducing repulsion between molecules.

Question 22

What happens to protein solubility at high salt concentrations?

Answer 22

Solubility decreases

Question 23

What happens to protein solubility at low salt concentrations?

Answer 23

Solubility increases (salting-in effect).

Question 24

What is the purpose of the BCA assay?

Answer 24

To estimate protein concentration by forming a purple complex with Cu⁺.

Question 25

Why is two-dimensional electrophoresis useful?

Answer 25

It separates proteins more precisely by using both charge and size.