Lecture 20 - Methods of protein analysis I

Question 1

Gel electrophoresis can be used to

Answer 1

determine the relative amount of different proteins in a sample

Question 2

In gel electrophoresis, small molecules

Answer 2

readily move through the porous gel

Question 3

In gel electrophoresis, proteins migrate from the

Answer 3

negative to the positive electrodes

Question 4

SDS-PAGE stands for

Answer 4

sodium dodecyl sulfate polyacrylamide gel electrophoresis

Question 5

A discontinuous buffer system contains (3)

Answer 5

1. Tank buffer (Tris-Gly pH 8.3)
2. Stacking buffer (Tris-HCl pH 6.8)
3. Resolving buffer (Tris-HCl pH 8.8)

Question 6

A stacking gel forces

Answer 6

the proteins to organize into a narrow band at the start of the resolving gel

Question 7

In SDS-PAGE, proteins that differ by in mass by about ____ can be distinguished

Answer 7

2%

Question 8

SDS-PAGE can be used to examine the efficacy of a protein purification protocol by

Answer 8

analyzing each fraction during purification and seeing the bands present

Question 9

Isoelectric focusing is a method that is used to separate proteins based on

Answer 9

their isoelectric point

Question 10

In isoelectric focusing, a protein stops moving in the gel when

Answer 10

it reaches the pH in the gel that is equal to its pI

Question 11

Protein concentration can be determined by (4)

Answer 11

1. UV absorption
2. Lowry (Folin-Ciocalteau) method
3. Bradford protein assay
4. Bicinchoninic Acid (BCA) method

Question 12

To follow the purification of a protein, we must also know the

Answer 12

enzyme activity in our sample

Question 13

Knowing the enzyme activity and the protein concentration, we can calculate the

Answer 13

specific activity

Question 14

Specific activity is the ratio of

Answer 14

enzyme activity to protein concentration

Question 15

Enzyme activity will ________ as the purification proceeds

Answer 15

decrease

Question 16

During purification, the specific activity of the protein will

Answer 16

increase

Question 17

The high specificity of antibodies for their target proteins allows us to (3)

Answer 17

1. Tag a specific protein
2. Make drugs
3. Image molecular targets in the body

Question 18

An antibody (also called an immunoglobulin, Ig) is

Answer 18

a protein, which is synthesized by animals in response to the presence of a foreign substance (antigen)

Question 19

Antibodies have specific and high affinity for

Answer 19

the antigens that elicited their synthesis

Question 20

An antibody recognizes

Answer 20

a specific group or cluster of amino acids on the target molecule called an antigenic determinant or epitope

Question 21

The specificity of the antibody-antigen interaction is a consequence of the

Answer 21

shape complementarity between the two surfaces

Question 22

Immunological methods depend on the ability to generate

Answer 22

antibodies to a specific antigen

Question 23

Polyclonal antibodies are derived from

Answer 23

multiple antibody-producing cell populations

Question 24

Most antigens have _______ epitopes

Answer 24

several

Question 25

Polyclonal antibodies are

Answer 25

heterogeneous mixtures of antibodies, each specific for one of the various epitopes on an antigen

Question 26

Monoclonal antibodies are

Answer 26

all identical, produced by clones of a single antibody-producing cell and recognize one specific epitope

Question 27

Is it usually more advantageous to work with polyclonal or monoclonal antibodies?

Answer 27

monoclonal

Question 28

ELISA stands for

Answer 28

enzyme-linked immunosorbent assay

Question 29

ELISA is used to

Answer 29

detect the amount of protein or other antigen present in a biological sample

Question 30

Indirect ELISA is used to

Answer 30

detect the presence of antibody

Question 31

Sandwich ELISA is used to

Answer 31

detect antigen rather than antibody

Question 32

Western blotting allows for detection of

Answer 32

a protein in a complex mixture

Question 33

In western blotting, antibodies can be labeled with (3)

Answer 33

1. Radioactive label
2. Fluorescent label
3. Enzyme that generates a colored product

Question 34

Cells can be stained with fluorescence-labeled antibodies to reveal

Answer 34

the location of a protein of interest

Question 35

Immunoprecipitation (IP) used

Answer 35

antibodies to “pull” a protein out of a solution