Lecture 7 Chapter 5 Techniques in Protein Biochemistry

Question 1

Sedimentation of a particle isn’t affected by:
a. mass.
b. shape.
c. the density of the solution.
d. charge.
e. size.

Answer 1

d. charge.

Question 2

Genome rather than proteome:
a. is a fixed characteristic of the cell.
b. provides a list of gene products that are present in an organism.
c. varies with the cell type and developmental stage.
d. is influenced by the presence of hormones or inhibitors, or other environmental conditions.
e. is the level of functional information, which encompasses the types, functions, and interactions of proteins that yield a functional unit.

Answer 2

a. is a fixed characteristic of the cell.

Question 3

The technique that obtains the isolation of the protein of interest from the thousands of other proteins in the cell is called:
a. salting out.
b. purification.
c. precipitation.
d. dialysis.
e. exclusion.

Answer 3

b. purification.

Question 4

What technique significantly increases the resolving power of protein purification?
a. salting out
b. gel electrophoresis
c. ion-exchange chromatography
d. high-pressure liquid chromatography
e. affinity chromatography

Answer 4

d. high-pressure liquid chromatography

Question 5

Choose the CORRECT definition of the given terms.
a. The total amount of protein is the measure of enzyme activity.
b. Enzyme activity is the ratio of total activity to the amount of protein in the enzyme assay.
c. An assay is not always based on some unique biochemical properties of the protein of interest.
d. The purification level is obtained by dividing specific activity of the initial extract by the total volume of the fraction.
e. The total activity is obtained by measuring specific activity of the fraction used in the assay and multiplying it by the fraction’s total volume.

Answer 5

b. Enzyme activity is the ratio of total activity to the amount of protein in the enzyme assay.

Question 6

What properties rise upon protein purification?
a. enzyme activity
b. amount of total protein
c. specific activity
d. bands on the gel electrophoresis
e. total activity

Answer 6

c. specific activity

Question 7

A mixture of all components of the cell that do NOT contain intact cells is called:
a. a pellet.
b. supernatant.
c. dialysate.
d. centrifugate.
e. homogenate.

Answer 7

e. homogenate.

Question 8

. If you want to get several fractions of decreasing density, each still containing hundreds of different proteins, in a step-by-step fashion what technique would you use?
a. gradient centrifugation
b. differential centrifugation
c. western blotting
d. gel electrophoresis
e. one of the chromatography methods

Answer 8

b. differential centrifugation

Question 9

What fraction would you get as a pellet after centrifugation of the tissue homogenate at 104,000 g for 64 minutes?
a. nuclear fraction
b. mitochondrial fraction
c. cytosol
d. microsomal fraction
e. cell membranes

Answer 9

c. cytosol

Question 10

Select all that apply. An intermediate product of protein purification is:
a. crude supernatant extract.
b. crude pellet extract.
c. homogenate.
d. supernatant extract.
e. dialysate.

Answer 10

b. crude pellet extract./d. supernatant extract.

Question 11

What technique is used to remove salt from a solution?
a. gel electrophoresis
b. salting out
c. dialysis
d. centrifugation
e. fractioning

Answer 11

c. dialysis

Question 12

Proteins with different sedimentation coefficients can be separated by:
a. gradient centrifugation.
b. differential centrifugation.
c. two-dimensional electrophoresis.
d. affinity chromatography.
e. ELISA.

Answer 12

b. differential centrifugation.

Question 13

The protein in a solution with a high concentration of (NH4)2SO4 doesn’t bind to a column. What technique should you use to solve this problem?
a. SDS-PAGE
b. ELISA
c. centrifugation
d. dialysis
e. HPLC

Answer 13

d. dialysis

Question 14

In which order will proteins A (55 kDa), B (60 Da), and C (50,000 Da) emerge from the column upon molecular exclusion chromatography?
a. A → B → C
b. A → C → B
c. B → A → C
d. B → C → A
e. C → A → B

Answer 14

b. A → C → B

Question 15

How can a protein that is positively charged at pH 7 be eluted in ion-exchange chromatography?
a. displacement of the protein by another ligand
b. increasing volume of the buffer
c. increasing pH of the buffer
d. increasing concentration of salt in the buffer
e. decreasing concentration of salt in the buffer

Answer 15

d. increasing concentration of salt in the buffer

Question 15

What technique is used to separate proteins by their net charge?
a. molecular exclusion chromatography
b. ion-exchange chromatography
c. two-dimensional electrophoresis
d. ELISA
e. immunoprecipitation

Answer 15

b. ion-exchange chromatography

Question 16

What amino acids should be prevailing in a protein at pH 7 for binding of that protein to beads containing negatively charged carboxylate groups?
a. serine
b. phenylalanine
c. histidine
d. glycine
e. cysteine

Answer 16

c. histidine

Question 17

Molecular exclusion chromatography is a technique of protein separation according to:
a. size.
b. isoelectric point.
c. binding affinity.
d. charge.
e. definition of individual proteins.

Answer 17

a. size.

Question 18

What is affinity chromatography based on?
a. separation of proteins by size
b. moving a molecule with a net charge in an electric field
c. affinity of proteins for specific chemical groups or specific molecules
d. separation of proteins by their net charge
e. high affinity of the antibody for the proteins

Answer 18

c. affinity of proteins for specific chemical groups or specific molecules

Question 19

What is ion-exchange chromatography based on?
a. separation of proteins by size
b. moving a molecule with a net charge in an electric field
c. affinity of proteins for specific chemical groups or specific molecules
d. separation of proteins by their net charge
e. high affinity of the antibody for the proteins

Answer 19

d. separation of proteins by their net charge

Question 20

. The largest number of individual proteins from a mixture is separated by:
a. affinity chromatography.
b. molecular exclusion chromatography.
c. ion-exchange chromatography.
d. gel filtration.
e. high-pressure liquid chromatography.

Answer 20

e. high-pressure liquid chromatography.

Question 21

What technique is used to separate proteins by their charge and then by their size?
a. molecular exclusion chromatography
b. ion-exchange chromatography
c. two-dimensional electrophoresis
d. ELISA
e. immunoprecipitation

Answer 21

c. two-dimensional electrophoresis

Question 22

Choose the FALSE statement about protein electrophoresis.
a. The gel serves as a molecular sieve that enhances separation.
b. The distance and speed that a protein moves depend on its shape.
c. Small proteins move rapidly through the gel.
d. The proteins in the gel can be visualized by staining them with silver.
e. It is performed in a thin, horizontal slab of polyacrylamide.

Answer 22

e. It is performed in a thin, horizontal slab of polyacrylamide.

Question 23

Separation of proteins only by their molecular weight by adding many negative charges to them is allowed by:
a. the flow from the cathode to the anode.
b. electricity.
c. adjusted pH.
d. SDS.
e. β-mercaptoethanol.

Answer 23

d. SDS.

Question 24

Protein purification can be monitored by measuring:
a. specific activity and performing SDS-PAGE.
b. total activity and performing SDS-PAGE.
c. total protein and performing HPLC.
d. specific activity and performing two-dimensional electrophoresis.
e. total activity and performing HPLC.

Answer 24

a. specific activity and performing SDS-PAGE.

Question 25

For enzymes, the assay is a measure of:
a. total protein.
b. total enzyme.
c. enzyme activity.
d. specific activity.
e. total activity.

Answer 25

c. enzyme activity.

Question 26

Differential centrifugation rather than gradient centrifugation:
a. is referred to as ultracentrifugation.
b. doesn’t depend of the shape of the particle.
c. requires the formation of a density gradient in a centrifuge tube.
d. can use radioactivity marks to determine the receptor from the fraction.
e. applies the harvesting of the separated bands of protein, collecting them drop by drop.

Answer 26

b. doesn’t depend of the shape of the particle.

Question 27

Choose the CORRECT statement.
a. The binding between Ig and antigen is a step in the immune response.
b. Epitope is composed of heavy and light chains.
c. Antigen is synthesized by an animal in response to the presence of a foreign substance.
d. The Fc domain of the Ig is formed by two heavy chains and two flexible linkers.
e. Nucleic acids can’t be effective antigens.

Answer 27

a. The binding between Ig and antigen is a step in the immune response.

Question 27

What reagent helps to visualize amino acids in amino acid composition analysis?
a. phenyl isothiocyanate
b. clostripain
c. β-mercaptoethanol
d. hydroxylamine
e. fluorescamine

Answer 27

e. fluorescamine

Question 28

Which statements describe sandwich ELISA? Select all that apply.
a. First, the antibody to a particular antigen is adsorbed to the bottom of a well.
b. The production of color indicates the amount of an antibody to a specific antigen.
c. This technique is used in tests for HIV infection.
d. The detection antibody with an enzyme linked to it binds to a specific antibody.
e. It is a quantitative method that permits the measurement of small quantities of an antigen.

Answer 28

a. First, the antibody to a particular antigen is adsorbed to the bottom of a well.
e. It is a quantitative method that permits the measurement of small quantities of an antigen.

Question 28

Polyclonal antibodies rather than monoclonal antibodies:
a. can be obtained by fusing of antibody-producing cells and myeloma cells.
b. are generated from a large number of cells of a single kind.
c. are used in purification of receptors or other proteins by immunoprecipitation.
d. such as Trastuzumab are used to treat some forms of breast cancer.
e. have an advantage for the detection of a protein with low abundance.

Answer 28

e. have an advantage for the detection of a protein with low abundance.

Question 28

Select all that apply. Enzyme-linked immunosorbent assay:
a. uses an enzyme, noncovalently linked to a specific antibody, that reacts with a colorless substrate to produce a colored product.
b. is used in tests for HIV infection.
c. is devoted to only two types.
d. can detect less than 10–9 g of a protein.
e. can be performed only with polyclonal antibodies.

Answer 28

a. uses an enzyme, noncovalently linked to a specific antibody, that reacts with a colorless substrate to produce a colored product.
c. is devoted to only two types.
e. can be performed only with polyclonal antibodies.

Question 29

. What technique allows the detection of very small quantities of a particular protein in a cell or in body fluid and makes it possible to find a protein in a complex mixture, which is useful in monitoring protein purification and in the cloning of genes?
a. sandwich ELISA
b. indirect ELISA
c. gradient centrifugation
d. immunoblotting
e. MALDI-TOF

Answer 29

d. immunoblotting

Question 30

What technique helps to determine amino acid sequence by removing one amino acid at a time from the amino end of a peptide?
a. Edman degradation
b. specific cleavage
c. peptides overlapping
d. electrospray ionization
e. MALDI-TOF

Answer 30

a. Edman degradation

Question 31

What is the most common protease used in sample preparation for a MALDI-TOF assay?
a. pepsin
b. papain
c. trypsin
d. thrombin
e. carboxypeptidase A

Answer 31

c. trypsin

Question 31

Which statement about mass spectrometry is FALSE?
a. In electrospray ionization, the protein or peptide under study is co-precipitated with an organic compound that absorbs laser light of an appropriate wavelength.
b. In the time of flight analysis, tiny amounts of biomolecules as small as a few picomoles (pmol) or femtomoles (fmol) can be analyzed.
c. In tandem mass spectrometry, peptides can be fragmented by bombardment with argon to generate a family of product ions in which one or more amino acids are removed from one end of the initial peptide analyte.
d. MALDI-TOF is one of the most accurate means of determining protein mass.
e. In MALDI-TOF, peptide masses are matched in a database against proteins that have been “electronically cleaved” by a computer simulating the same fragmentation technique used for the experimental sample.

Answer 31

a. In electrospray ionization, the protein or peptide under study is co-precipitated with an organic compound that absorbs laser light of an appropriate wavelength.

Question 32

. What reagent cleaves the carboxyl side of lysine and arginine residues in proteins?
a. hydroxylamine
b. staphylococcal protease
c. clostripain
d. trypsin
e. carboxypeptidase A

Answer 32

d. trypsin

Question 33

When enzymes are purified, the assay is often based on:
a. light absorbance.
b. catalytic activity.
c. pH.
d. temperature changes.
e. mRNA levels.

Answer 33

b. catalytic activity.

Question 34

Two-dimensional electrophoresis is a combination of what two techniques?
a. isoelectric focusing and affinity chromatography
b. ion-exchange chromatography and SDS-PAGE
c. affinity chromatography and SDS-PAGE
d. isoelectric focusing and SDS-PAGE
e. isoelectric focusing and ion-exchange chromatography

Answer 34

d. isoelectric focusing and SDS-PAGE

Question 35

What technique can be used to determine the size of a target protein?
a. Edman degradation
b. affinity chromatography
c. western blot
d. ELISA
e. isoelectric focusing gel

Answer 35

c. western blot

Question 36

Antibodies as reagents are used by:
a. western blotting.
b. gradient centrifugation.
c. gel electrophoresis.
d. ELISA.
e. MALDI-TOF.

Answer 36

d. ELISA.

Question 37

Which technique CANNOT be used for quantitative analysis?
a. gradient centrifugation
b. ELISA
c. enzyme assay
d. SDS-PAGE
e. chromatography

Answer 37

a. gradient centrifugation

Question 38

What is TRUE regarding gel filtration chromatography and PAGE?
a. In both, small proteins move most rapidly.
b. In both, large proteins move most rapidly.
c. In PAGE, large proteins move most rapidly but in gel filtration, small proteins move most rapidly.
d. In gel filtration, large proteins move most rapidly but in PAGE, small proteins move most rapidly.
e. In both, proteins are always in their native structure.

Answer 38

d. In gel filtration, large proteins move most rapidly but in PAGE, small proteins move most rapidly.

Question 39

Two proteins are similar in size but differ significantly in the number of acidic and basic amino acids. What technique would be BEST suited for separating these two proteins?
a. SDS-PAGE and gel-filtration chromatography
b. isoelectric focusing and dialysis
c. immunoprecipitation and affinity chromatography
d. isoelectric focusing and ion-exchange chromatography
e. SDS-PAGE and immunoprecipitation

Answer 39

d. isoelectric focusing and ion-exchange chromatography

Question 40

Two proteins are similar in the number of acidic and basic amino acids but significantly differ in size. What technique would be BEST suited for separating these two proteins?
a. SDS-PAGE and gel-filtration chromatography
b. isoelectric focusing and dialysis
c. immunoprecipitation and affinity chromatography
d. isoelectric focusing and ion-exchange chromatography
e. isoelectric focusing and affinity chromatography

Answer 40

a. SDS-PAGE and gel-filtration chromatography

Question 41

Calmodulin is a calcium-binding protein expressed in eukaryotic cells. What two techniques would greatly reduce the number of steps to purify calmodulin?
a. SDS-PAGE and gel-filtration chromatography
b. isoelectric focusing and dialysis
c. immunoprecipitation and affinity chromatography
d. isoelectric focusing and ion-exchange chromatography
e. immunoprecipitation and ion-exchange chromatography

Answer 41

c. immunoprecipitation and affinity chromatography

Question 42

Select all that apply. In isoelectric focusing:
a. proteins migrate to the location at which they have no net charge.
b. the pH of a gel depends on the isoelectric point of the protein.
c. a gel for isoelectric focusing contains SDS.
d. a mixture of proteins with the same isoelectric point is subjected to electrophoresis.
e. the isoelectric point (pI) of a protein is the pH at which its net charge is zero.

Answer 42

a. proteins migrate to the location at which they have no net charge.
e. the isoelectric point (pI) of a protein is the pH at which its net charge is zero.

Question 43

You are interested in studying a powerful enzyme that is expressed in low amounts. What technique would you choose to determine how much of it is found in the tissue of interest?
a. MALDI-TOF mass spectrometry to determine the protein mass
b. ELISA to identify any antigenic determinants
c. Amino Acid Composition Analysis because it can be done for the whole protein
d. two-dimensional electrophoresis to determine charge and size data of the protein
e. salting out to concentrate the protein for further study

Answer 43

b. ELISA to identify any antigenic determinants

Question 44

What is the term for the subset of gene products actually expressed by a cell?
a. genomics
b. metabolome
c. genome
d. Svedberg
e. proteome

Answer 44

e. proteome

Question 45

The sedimentation coefficient is expressed in _______ units.

Answer 45

ANSWER: Svedberg