Deck 5: Methods in Protein Biochemistry

Question 1

The following peptides are separated using an anion exchange resin in ion-exchange chromatography. Which peptide is eluted first?

A) A
B) B
C) C
D) D

Answer 1

D

Question 2

Which peptide would migrate the slowest in an SDS-PAGE gel?

A) A
B) B
C) C
D) D

Answer 2

C

Question 3

Which percentage of polyacrylamide would give the best separation for large proteins?

A) 5%
B) 7.5%
C) 10%
D) 20%

Answer 3

5%

Question 4

Why is the process of purifying proteins from cells considered challenging?

A) Proteins are insoluble.
B) The process is expensive and time consuming.
C) There is no way to determine the structure of the protein.
D) There are 10,000 to 100,000 proteins in one sample.

Question 5

After centrifugation, there is a 10% decrease in activity and a 75% decrease in total protein. What is purification of the target protein?

A) 0.28-fold
B) 1.3-fold
C) 3.6-fold
D) 7.5-fold

Question 6

When preparing to isolate proteins from plant cells, the first step in preparing the cell homogenate would be

A) sonication.
B) using a French press.
C) treatment with mild detergents.
D) enzymatic treatment.

Question 7

Calculate the purification of the target protein when there is a 30% decrease in activity and a 55% decrease in total protein after centrifugation.

A) 0.55-fold
B) 0.64-fold
C) 1.6-fold
D) 1.8-fold

Question 8

Which peptide would migrate the fastest in a SDS-PAGE gel?

A) A
B) B
C) C
D) D

Question 9

The figure below shows three proteins that are separated using gel filtration chromatography. Which protein is the largest?

A) squares
B) circles
C) triangles
D) Not enough information is included to determine the protein.

Question 10

The figure below shows how differential centrifugation can be used to isolate four subcellular fractions. Which fraction corresponds to the cytosol fraction?

A) A
B) B
C) C
D) D

Question 11

Which separation technique exploits the solubility differences of proteins?

A) centrifugation
B) salting out
C) dialysis
D) ion-exchange chromatography

Question 12

After centrifugation, the purity of the protein is determined by

A) absorbance measurements.
B) activity units.
C) total protein content.
D) specific activity.

Question 13

Calculate the specific activity when 500 mg of protein has an activity of 18,000 units.

A) 0.28 units/mg protein
B) 36 units/mg protein
C) 9000 units/mg protein
D) 13,000 units/mg protein

Question 14

Which percentage of polyacrylamide would give the best separation for small proteins?

A) 5%
B) 7.5%
C) 10%
D) 20%

Question 15

The figure below shows how differential centrifugation can be used to isolate four subcellular fractions. Which fraction corresponds to the membrane fraction?

A) A
B) B
C) C
D) D

Question 16

Which type of chromatography separates proteins using specific binding properties?

A) affinity chromatography
B) gel filtration chromatography
C) size exclusion chromatography
D) high-performance liquid chromatography

Question 17

Which of the following peptides would be eluted last when separated using gel filtration chromatography?

A) A
B) B
C) C
D) D

Question 18

The figure below shows how differential centrifugation can be used to isolate four subcellular fractions. Which fraction corresponds to the mitochondrial fraction?

A) A
B) B
C) C
D) D

Question 19

Biochemical assays are important in the process of isolating proteins because they

A) uniquely identify one protein.
B) separate proteins based on polarity.
C) isolate proteins based on size.
D) determine the molecular weight of proteins.

Question 20

After using the salting out method to isolate proteins, how is ammonium sulfate removed from the solution?

A) sonication
B) French press
C) dialysis
D) centrifugation

Question 21

Two-dimensional polyacrylamide gel electrophoresis separates proteins based on

A) pI and shape.
B) ligand affinity and molecular weight.
C) shape and ligand affinity.
D) pI and molecular weight.

Question 22

In tandem mass spectrometry, the second mass spectrometer

A) determines the nuclear spin of the atoms.
B) determines the mass of peptide subfragments.
C) uses bioinformatics to compare the masses of the peptides.
D) uses electrospray ionization to select peptide fragments.

Question 23

In tandem mass spectrometry, the first mass spectrometer

A) determines the nuclear spin of the atoms.
B) determines the mass of peptide subfragments.
C) uses bioinformatics to compare the masses of the peptides.
D) uses electrospray ionization to select peptide fragments.

Question 24

Predict the fragments of the following peptide after cleavage by the V-8 protease. CMVAADGKLEPVS

A) CMVAAD GKLEPVS
B) CMVAADGKLE PVS
C) CMVAADG KLEPVS
D) CMVAAD GKLE PVS

Question 25

During solid-phase peptide synthesis, 9-fluorenylmethoxycarbonyl is used

A) as a blocking agent.
B) as an activating agent.
C) to cleave the peptide from the resin.
D) to couple amino acids during the synthesis.

Question 26

Which enzyme or reagent cleaves a peptide at the carboxyl side of a methionine residue?

A) trypsin
B) chymotrypsin
C) V-8 protease
D) cyanogen bromide

Question 27

The advantage of using an SDS-PAGE gel compared with a native PAGE gel is that the SDS-PAGE gel

A) separates proteins only based on molar mass.
B) gives information about the charge or conformation of the protein.
C) results in a better separation of small proteins.
D) increases the resolution of large and small proteins.

Question 28

The step in solid-phase peptide synthesis that occurs at number 2 is

A) deblocking of the residue attached to the resin.
B) coupling of the amino acid.
C) cleavage from the resin.
D) activation of the Fmoc blocked residue.

Question 29

Which method uses mass to charge ratios as well as a bioinformatics approach to determine the amino acid composition of a protein?

A) X-ray crystallography
B) nuclear magnetic resonance
C) tandem mass spectrometry
D) size exclusion chromatography

Question 30

In solid-phase peptide synthesis, which reagent is used to remove the peptide from the solid resin support?

A) HF
B) DCC
C) Fmoc
D) PITC

Question 31

The step in solid-phase peptide synthesis that occurs at number 3 is

A) deblocking of the residue attached to the resin.
B) coupling of the amino acid.
C) cleavage from the resin.
D) activation of the Fmoc blocked residue.

Question 32

In isoelectric focusing, a protein with a pH below the pI would

A) migrate toward the anode.
B) migrate toward the cathode.
C) stop migrating.
D) migrate, but there is no way to determine the direction.

Question 33

A polypeptide was digested by trypsin and chymotrypsin. Use the following information to determine the polypeptide sequence.

A) ECFGMREDWKYLM
B) LMYKWMGFCEDER
C) LMYKWDERMGFCE
D) CELMYKWDERMGF

Question 34

In isoelectric focusing,

A) negatively charged proteins migrate toward the cathode.
B) negatively charged proteins migrate toward the anode.
C) small proteins migrate the fastest.
D) large proteins migrate the fastest.

Question 35

In isoelectric focusing,

A) positively charged proteins migrate toward the cathode.
B) positively charged proteins migrate toward the anode.
C) small proteins migrate the fastest.
D) large proteins migrate the fastest.

Question 36

The advantage of using a native PAGE gel compared with an SDS-PAGE gel is that the native PAGE gel

A) separates proteins only based on molar mass.
B) gives information on the charge or conformation of the protein.
C) results in a better separation of small proteins.
D) increases the resolution of large and small proteins.

Question 37

Which technique ionizes polypeptides by embedding the tryptic fragments into a light-absorbing matrix and exposing it to a laser?

A) ESI
B) MALDI
C) NMR
D) X-ray crystallography

Question 38

Predict the fragments of the following peptide after cleavage by trypsin. GLMKTYPDSTA

A) GLM KTYPDSTA
B) GLMK TYPDSTA
C) GLMKTY PDSTA
D) GLMKTYPD STA

Question 39

Which technique ionizes polypeptides by releasing them from a small metallic capillary at high voltage?

A) ESI
B) MALDI
C) NMR
D) X-ray crystallography

Question 40

Below are the steps of solid-phase peptide synthesis: 1. The protecting groups of the amino acids are removed.
2) The peptide is released from the solid support resin.
3) An incoming amino acid is activated at the carboxyl group by DCC and added to the column.
4) Fmoc is removed by treatment with a base and the C-terminal amino acid is attached to the resin molecule.
"5) The resin bound C-terminal amino acid and the incoming N-terminal amino acid are coupled.
What is the appropriate order of these steps?"

A) 3, 5, 4, 2, 1
B) 1, 4, 2, 5, 3
C) 4, 3, 5, 1, 2
D) 2, 4, 5, 3, 1

Question 41

Protein NMR is more useful than X-ray crystallography for studying

A) secondary structure elements.
B) large proteins.
C) protein unfolding.
D) static protein structures.

Question 42

What is the advantage of using monoclonal antibodies compared with polyclonal antibodies?

A) They are less labor intensive to generate.
B) They can recognize more than one epitope.
C) The process used to produce the antibodies provides unlimited supplies.
D) They can be generated without using animals.

Question 43

The size of proteins studied by NMR is limited because

A) a small sample size is needed.
B) it is hard to determine the phases of diffracted X-rays.
C) the measurements of the distances between atoms are only approximate.
D) large molecules reorient slowly in solution.

Question 44

Using the figure below describing monoclonal antibody generation, identify the step where the B cells are fused with the tumor cells.

A) 1
B) 2
C) 3
D) 4

Question 45

Epitopes on protein antigens are generally located

A) in the heavy chain domains.
B) on polar surface groups in loop regions.
C) in the light chain domains.
D) in association with variable domain amino acid residues.

Question 46

Monoclonal antibodies

A) recognize a single epitope.
B) recognize multiple epitopes.
C) are isolated from leukocytes.
D) are isolated from red blood cells.

Question 47

During the production of polyclonal antibodies, where are the antibodies isolated from?

A) an immortalized tumor cell line
B) liver cells
C) spleen cells
D) blood cells

Question 48

What cells are shown at step number 5 in the figure below?

A) B cells
B) immortalized tumor cell
C) hybridoma cells
D) red blood cells

Question 49

The Fab immunoglobin fragment

A) is generally found within $\beta$ -barrels.
B) is located in polar surface groups in loop regions.
C) contains the high-affinity antigen binding site.
D) is responsible for the solubility of the antibody.

Question 50

During the production of polyclonal antibodies, how are the antigen-specific antibodies purified?

A) dialysis
B) gel electrophoresis
C) size exclusion chromatography
D) affinity chromatography

Question 51

When cells isolated from the spleen are fused with long-living cells, they produce

A) B cells.
B) immortalized tumor cells.
C) hybridoma cells.
D) red blood cells.

Question 52

The specific sites on the antigen that interacts with the antibody are called

A) epitopes.
B) immunoglobin light chains.
C) immunoglobin heavy chains.
D) variable-domain amino acid residues.

Question 53

Why is the supply of polyclonal antibodies limited?

A) They are time consuming to produce.
B) The yield is very low.
C) The animals the antibodies are isolated from eventually die.
D) The production is limited by the government.

Question 54

What cells are shown at step number 2 in the figure below?

A) B cells
B) immortalized tumor cells
C) hybridoma cells
D) red blood cells

Question 55

The majority of serum proteins and nonspecific antibodies are removed during the generation of polyclonal antibodies by

A) washing with a low pH buffer.
B) gel electrophoresis.
C) size exclusion chromatography.
D) salting out.

Question 56

During solid-phase peptide synthesis, dicyclohexylcarbodiimide is used

A) as a blocking agent.
B) as an activating agent.
C) to cleave the peptide from the resin.
D) to couple amino acids during the synthesis.

Question 57

What is the advantage of using polyclonal antibodies compared with monoclonal antibodies?

A) They are less labor intensive to generate.
B) They provide the ability to monitor quality continually.
C) The process used to produce the antibodies provides unlimited supplies.
D) They can be generated without using animals.

Question 58

One of the most difficult steps in X-ray crystallography is

A) using the correct radio frequency pulses to perturb the nuclear spin.
B) determining the phases of diffracted X-rays.
C) dissolving the protein in the appropriate solvent.
D) obtaining a large enough sample for analysis.

Question 59

Polyclonal antibodies are

A) derived from B cells isolated from the spleen.
B) isolated from hybridoma cells.
C) a heterogeneous mixture.
D) a homogenous mixture.

Question 60

The specific sites on the antibody that interacts with the antigen are

A) epitopes.
B) immunoglobin light chains.
C) immunoglobin heavy chains.
D) variable-domain amino acid residues.

Question 61

The part of the Western blot that contains the protein-specific recognition and facilitates the antigen-antibody interactions is the

A) SDS-PAGE gel.
B) primary antibody.
C) tertiary antibody.
D) filter membrane.

Question 62

Which animal is most often used to generate monoclonal antibodies?

A) rabbit
B) chicken
C) goat
D) mouse

Question 63

Co-immunoprecipitation proteins are separated through

A) affinity chromatography.
B) SDS-PAGE.
C) HPLC.
D) size exclusion chromatography.

Question 64

Below are the steps of a Western blot. What is the correct order? 1. The membrane is incubated with the secondary antibody.
2) The membrane is incubated with the primary antibody.
3) The membrane is washed with a blocking solution.
4) Proteins are transferred to a nitrocellulose membrane.
5) Protein extracts are separated by gel electrophoresis.

A) 5, 4, 3, 2, 1
B) 4, 5, 2, 3, 1
C) 3, 4, 5, 1, 2
D) 2, 5, 4, 3, 1

Question 65

Compare and contrast the three MOST commonly used homogenization techniques to prepare a cell extract.

Question 66

What is the advantage of using sodium dodecyl sulfate (SDS) in gel electrophoresis?

Question 67

In an ELISA, the capture antibody is a antibody.

A) monoclonal primary
B) polyclonal primary
C) monoclonal secondary
D) polyclonal secondary

Question 68

Most secondary antibodies used in Western blots are covalently linked to the enzyme

A) luciferase.
B) horseradish peroxidase.
C) catalase.
D) hydrolase.

Question 69

Explain how to separate the following proteins using two different types of chromatography.

Question 70

In an ELISA, the detection antibody is a antibody.

A) monoclonal primary
B) polyclonal tertiary
C) monoclonal secondary
D) polyclonal secondary

Question 71

Protein-specific antibodies can be used to detect proteins within cells conserving the cell architecture using

A) immunofluorescence.
B) Western blot.
C) immunoprecipitation.
D) ELISA.

Question 72

Immunoprecipitation uses an antibody-bead combination to

A) detect the antigen.
B) enrich the protein solution.
C) digest the proteins for use in mass spectrometry.
D) separate the proteins using centrifugation.

Question 73

During a Western blot, the membrane is washed with a blocking solution before being treated with the primary antibody to

A) retain the position of the proteins in the transfer from gel to membrane.
B) facilitate antibody-antigen interactions.
C) decrease nonspecific antibody binding.
D) facilitate a chemical reaction that can be detected.

Question 74

Explain the differences among the three types of column chromatography.

Question 75

Which technique can be combined with mass spectrometry to identify protein antigens in large cellular complexes?

A) immunofluorescence
B) Western blot
C) immunoprecipitation
D) ELISA

Question 76

Which technique uses highly specific monoclonal antibodies in a high-throughput form to detect small amounts of antigen?

A) immunofluorescence
B) Western blot
C) SDS-PAGE gel
D) ELISA

Question 77

If a protocol for a Western blot calls for the use of rabbit anti-mouse antibody, what type of antibody would this be considered?

A) monoclonal primary
B) polyclonal primary
C) monoclonal secondary
D) polyclonal secondary

Question 78

In monoclonal antibody generation, the cells that produce the antibody in the animal are located in cells.

A) heart
B) liver
C) spleen
D) red blood

Question 79

Why is protein purification often referred to as an art and a science?

Question 80

Which component used in a Western blot is covalently linked to an enzyme?

A) SDS-PAGE gel
B) primary antibody
C) secondary antibody
D) filter membrane