Deck 5: Protein Purification and Characterization Techniques

Question 1

The following methods are useful for cell homogenization:

A) Sonication.
B) Freezing and thawing.
C) Detergents.
D) Enzymes.
E) All of these are correct.

Answer 1

All of these are correct.

Question 2

The purity of an enzyme at various stages of purification is best measured by

A) total protein.
B) total enzyme activity.
C) specific activity of the enzyme.
D) percent recovery of the protein.
E) percent recovery of the enzyme.

Answer 2

specific activity of the enzyme.

Question 3

The typical order for the major steps of enzyme isolation would be (from first to last):

A) Homogenization, salt fractionation, electrophoresis, column chromatography.
B) Homogenization, column chromatography, salt fractionation, electrophoresis.
C) Homogenization, salt fractionation, column chromatography, electrophoresis.
D) Salt fractionation, homogenization, electrophoresis, column chromatography.
E) Homogenization, electrophoresis, salt fractionation, column chromatography.

Answer 3

Homogenization, salt fractionation, column chromatography, electrophoresis.

Question 4

Which of the following are principles on which to base column chromatography?

A) Molecular size
B) Isoionic pH or pI
C) Ion exchange
D) Both molecular size and ion exchange
E) All of these

Question 5

Which separates based on the ionic charge on a protein?

A) Gel filtration
B) Affinity chromatography
C) Cation exchange
D) Anion exchange
E) Cation or anion exchange

Question 6

In gel filtration chromatography

A) materials are separated based on their size, the smaller ones eluting first.
B) materials are separated based on their size, the larger ones eluting first.
C) materials are separated based on their hydrophobic nature, the more hydrophobic ones eluting first.
D) materials are separated based on their hydrophobic nature, the less hydrophobic ones eluting first.

Question 7

Differential centrifugation at low speeds (several thousand RPM) is a useful step when

A) organelles have been lysed.
B) enzymes of interest have different sizes.
C) cell membranes must be left intact.
D) ribosomes need to be broken down.
E) there are either organelles or debris to separate.

Question 8

Using differential centrifugation it is possible to separate

A) nuclei, mitochondria, and ribosomes into three separate fractions
B) organelles from contaminating salts
C) proteins that differ in charge
D) proteins from membranes

Question 9

Which would be best to separate proteins of similar size?

A) Gel filtration
B) Affinity chromatography
C) Cation exchange
D) Anion exchange
E) Cation or anion exchange

Question 10

Which of the following happens as a protein is purified?

A) the percent recovery and the fold purification both increase
B) the percent recovery and the fold purification both decrease
C) the percent recovery increases and the fold purification decreases
D) the percent recovery decreases and the fold purification increases

Question 11

In affinity chromatography, a protein

A) which binds to the ligand will remain on the column.
B) which binds to the ligand will elute from the column.
C) which is hydrophobic will remain on the column.
D) which is hydrophilic will remain on the column.

Question 12

Which type of column is most affected by the shape of the protein, for example, comparing spherical and cigar-shaped proteins?

A) Gel filtration
B) Affinity chromatography
C) Cation exchange
D) Anion exchange
E) Cation or anion exchange

Question 13

Ammonium sulfate is useful in protein purification because

A) it contains nitrogen and sulfur, both of which occur in proteins
B) it is sparingly soluble in water, causing proteins to co-precipitate with it
C) very pure proteins are obtained when it is used
D) it forms ion-dipole interactions with water, making proteins less soluble and more likely to precipitate

Question 14

Elution of proteins by means of a pH gradient would work best with this type of column:

A) Gel filtration
B) Affinity chromatography
C) Cation exchange
D) Anion exchange
E) Cation or anion exchange

Question 15

Which would be best to separate a protein that binds strongly to its substrate?

A) Gel filtration
B) Affinity chromatography
C) Cation exchange
D) Anion exchange
E) Cation or anion exchange

Question 16

The typical order of differential centrifugation for organelles is (from slowest speed\lowest g to fastest speed\highest g ):

A) nuclei, microsomes, mitochondria & chloroplasts, cytosol, whole cells
B) whole cells, nuclei, mitochondria & chloroplasts, microsomes, cytosol
C) cytosol, microsomes, nuclei, mitochondria & chloroplasts, whole cells
D) nuclei, mitochondria & chloroplasts, whole cells, cytosol, microsomes
E) whole cells, cytosol, microsomes, nuclei, mitochondria & chloroplasts

Question 17

What tends to happen to the percent recovery during a protein's purification?

A) The number usually steadily increases during the purification.
B) The number usually steadily decreases during the purification.
C) The number usually stays fairly constant during the purification.
D) There is no general trend for percent recovery during a protein purification.

Question 18

Salting out with ammonium sulfate is based upon proteins interacting with other proteins via

A) hydrogen bonds.
B) ionic bonds.
C) hydrophobic interactions.
D) disulfide bonds.

Question 19

Which would be best to separate positively charged proteins?

A) Gel filtration
B) Affinity chromatography
C) Cation exchange
D) Anion exchange
E) Cation or anion exchange

Question 20

Which separates on the basis of molecular weight?

A) Gel filtration
B) Affinity chromatography
C) Cation exchange
D) Anion exchange
E) Cation or anion exchange

Question 21

The degree of separation in molecular sieve chromatography depends on

A) the polarity of the mobile phase
B) the pK_a of the buffer material in the mobile phase
C) the chemical nature of the sieve material
D) the size of the pores in the sieve material

Question 22

A separation of a mixture of cations of different charge requires

A) another cationic substance
B) an anionic substance
C) an electrically neutral, but highly polar, substance
D) an electrically neutral, nonpolar substance

Question 23

Which substance would you expect to be eluted first from a molecular sieve column with a suitable degree of crosslinking?

A) hemoglobin
B) myoglobin
C) 2,3- bis phosphoglycerate
D) all would elute at the same rate

Question 24

In the SDS-PAGE (sodium dodecylsulfate - polyacrylamide gel electrophoresis) method, separation takes place on the basis of

A) charge only, because all particles have different charges, but the same mass.
B) the sieving action of the gel, because all particles have the same charge, but different masses.
C) the sieving action of the gel, because all particles have approximately the same charge\mass ratio, but different masses.
D) the chemical nature of the buffer used as the electrolyte.

Question 25

The order of elution of AAs H, E, & K from a cation exchange column by a pH 6 buffer is

A) H E K
B) E H K
C) K H E
D) E K H

Question 26

Exhibit 5A The following diagram shows the anode, cathode, and pH gradient of an isoelectric focusing bed: Refer to Exhibit 5A. If the amino acid glycine were placed in the bed where the pH is 7, and the current were turned on, it would migrate closest to which of the following positions?

A) pH 4
B) pH 6
C) pH 8
D) pH 10

Question 27

The following are all principles on which to base electrophoresis except:

A) Molecular size
B) Isoionic pH or pI
C) Net charge
D) Binding to a substrate
E) Shape

Question 28

Exhibit 5A The following diagram shows the anode, cathode, and pH gradient of an isoelectric focusing bed: Refer to Exhibit 5A. A mixture of asp, asn, and arg is placed in the bed where the pH is 7, and the current is turned on. From left to right, which best represents the final positions of the individual amino acids?

A) asp asn arg
B) arg asn asp
C) asn asp arg
D) arg asp asn

Question 29

Exhibit 5A The following diagram shows the anode, cathode, and pH gradient of an isoelectric focusing bed: Refer to Exhibit 5A. If the amino acid glycine were placed in the bed where the pH is 11, and the current were turned on, it would migrate closest to which of the following positions?

A) pH 4
B) pH 6
C) pH 8
D) pH 10

Question 30

Which of the following is not an example of column chromatography?

A) ammonium sulfate fractionation
B) ion-exchange separation
C) HPLC
D) affinity separation

Question 31

In all forms of chromatography one way of identifying eluted substances is by

A) fluorescence spectroscopy
B) comparison with standards
C) radioactive labeling
D) treating fractions with a reagent that will cause a color change

Question 32

In a sample consisting of lysine, leucine, and glutamic acid, which will be eluted last from an anion exchange resin at pH 7?

A) all three will be eluted at the same time
B) lysine
C) leucine
D) glutamic acid

Question 33

In any form of chromatography, how will a compound which interacts more strongly with the stationary phase elute compared to one that interacts less strongly?

A) A compound interacting more strongly will elute earlier than one with weaker interactions.
B) A compound interacting more strongly will elute later than one with weaker interactions.
C) The order of elution has nothing to do with interactions with the stationary phase, but with interactions with the mobile phase.

Question 34

When electrophoretic separations are done based on molecular weight, the distance that a molecule moves can be graphed as a straight line when compared to:

A) the MW of the proteins
B) the negative of the MW of the proteins
C) the log of the MW of the proteins
D) none of these

Question 35

In electrophoresis experiments

A) the separation must be carried out in bright light
B) the polarity of substances to be separated is more important than their charge or size
C) the sample can be badly degraded as a result of the separation
D) an electric field must be applied to the mixture to be separated

Question 36

In affinity chromatography

A) there is nonspecific binding of proteins to column material
B) only minor purifications can be obtained
C) the mobile phase is always pure water
D) the ligand is always specific for one type of protein to be bound
E) there can be molecule specific ligands or group specific ligands

Question 37

In chromatography the experimental setup always requires

A) a stationary phase and a mobile phase
B) a spectrophotometric detecting device
C) a sample in which components differ in charge
D) a sample in which components differ in polarity

Question 38

How many bands would be produced when hemoglobin is subjected to SDS-PAGE?

A) 1
B) 2
C) 3
D) 4
E) 2, but only if the size of the pores in the gel would allow two proteins of slightly different size to be separated

Question 39

Two-dimensional electrophoresis usually exploits these 2 different properties of proteins:

A) Molecular weight and shape
B) Molecular weight and net charge
C) Molecular weight and isoionic pH
D) Isoionic pH and shape
E) Isoionic pH and net charge

Question 40

A chromatography technique where a solution of nonpolar compounds is put through a column that has a nonpolar liquid immobilized on an inert matrix is which type of chromatography?

A) Gel filtration
B) Ion exchange
C) Affinity
D) HPLC
E) Reverse Phase HPLC

Question 41

If a protein with the sequence PQRKYPIG is treated with trypsin, what will the products be?

A) PQR KYPIG
B) PQRK YPIG
C) PQR K YPIG
D) PQ R KPIG0

Question 42

The most efficient method for determining the sequence of a short peptide is:

A) Edman degradation
B) Trypsin digestion
C) Chymotrypsin digestion
D) Cyanogen bromide digestion

Question 43

Determination of the sequence of amino acids in a peptide is done by

A) x-ray crystallography
B) Edman degradation
C) treatment with cyanogen bromide
D) trypsin hydrolysis

Question 44

When end-group analysis was done on the protein insulin, the results indicated that both glycine and phenylalanine were N-terminal amino acids and both asparagine and alanine were C-terminal amino acids. These results indicate that

A) the experiment was done incorrectly
B) no conclusions can be drawn
C) there were impurities in the sample
D) insulin consists of two polypeptide chains

Question 45

Important steps in sequencing pure proteins include all of these except:

A) Determining the amino acid composition
B) Determining the isoionic pH of the protein
C) Breaking the protein into smaller peptides
D) Determining the amino acids on the ends of the protein
E) Determining the amino acids on the ends of the smaller peptides

Question 46

Cyanogen bromide (CNBr) cleaves proteins

A) after positively charged residues, such as K & R.
B) after negatively charged residues, such as D & E.
C) after aromatic residues, such as Y & W.
D) after methionine residues.

Question 47

It is impossible to sequence a protein if you do not have overlapping sequences to work with.

Question 48

In isoelectric focusing gel electrophoresis

A) particular care must be taken to ensure the same pH along the length of the gel
B) there is a pH gradient that parallels the electric field gradient
C) the electric current is allowed to fluctuate
D) the electric circuits of the apparatus must be very well insulated

Question 49

The isoelectric point is

A) the pH at which a substance has no net charge
B) the pH at which a substance has a net positive charge
C) the pH at which a substance has a net negative charge
D) the pH at which a substance has no charge groups of any kind

Question 50

An amino acid analyzer is an instrument used to determine

A) the sequence of amino acids in a polypeptide chain
B) the identity of N-terminal and C-terminal amino acids in a protein
C) the presence of modified amino acids in a protein
D) the identities and relative amounts of amino acids in a protein

Question 51

In an ELISA, the molecule that binds directly to the target protein is called the

A) primary antibody
B) secondary antibody
C) linked enzyme
D) immunosorbent

Question 52

If a protein with the sequence FEWPRQVDMARINE is treated with chymotrypsin, what will the products be?

A) F EW PRQVMARINE
B) FE WPRQVD MARINE
C) FEWPR QVDMAR INE
D) FEWPRQVDM ARINE

Question 53

It is frequently possible to bypass the determination of the identity of the N-terminal amino acid of a protein because

A) this information is already available from the amino acid analysis
B) the Edman method sequences the peptide from the N-terminal end
C) N-terminal amino acids are always chemically modified
D) this information is not needed

Question 54

Which of the following explains why a secondary antibody is used in an ELISA?

A) two antibodies will be twice as visible as one antibody
B) it allows the visible signal to be amplified
C) primary antibodies cannot be tagged with visible markers
D) none of the choices

Question 55

Two dimensional separation methods

A) lead to unreliable results
B) are not widely used because of their complexity
C) do not improve separation
D) consist of two separation methods applied in sequence

Question 56

Methods for breaking proteins into smaller peptides include all of the following except:

A) Digestion with chymotrypsin
B) Cyanogen Bromide treatment
C) Digestion with Trypsin
D) Edmann degradation
E) All of the above create short peptides suitable for sequencing

Question 57

Matrix-Assisted Laser Desorption Ionization is a type of _____ technique.

A) electrophoresis
B) ion exchange chromatography
C) affinity chromatography
D) mass spectrometry

Question 58

Which of the following treatments results in a polypeptide fragment with a homoserine lactone at the C-terminal end?

A) trypsin
B) chymotrypsin
C) cyanogen bromide
D) Edman method

Question 59

Generally speaking, sequence techniques have become so sensitive that if you are able to isolate the protein on a gel, there is enough of it to get a significant amount of its sequence.

Question 60

Which of the following is not used in protein structure determination?

A) digestion with proteolytic enzymes
B) the Edman method
C) treatment with cyanogen bromide
D) treatment with alkyl halides

Question 61

Which of the following is a similarity between ion-exchange chromatography and affinity chromatography?

A) Both use a column resin that binds the protein of interest.
B) Both involve a less-specific interaction and are based on net charge.
C) Both separate molecules of varying molecular weights.
D) Both use very high-resolution columns that can run under high pressures.

Question 62

Studying how proteins interact with other proteins is called

A) expression proteomics
B) structural proteomics
C) interaction proteomics
D) none of the choices

Question 63

The process of breaking cells open to release the organelles is called _____.

A) comminution
B) distillation
C) homogenization
D) liquefaction

Question 64

In an ELISA, the compound 4-chloro-1-naphthol is used because

A) it helps the secondary antibody to bind to the protein
B) it helps the primary antibody bind to the protein
C) it turns color in the presence of an enzyme that is bound to the secondary antibody
D) all of the choices

Question 65

What is the distinguishing feature of affinity chromatography?

Question 66

A protein chip is functionally most similar to which other technique?

A) gel filtration
B) ELISA
C) SDS-PAGE
D) western blot

Question 67

The most common way of visualizing the results seen on a protein chip is by use of

A) secondary antibodies
B) primary antibodies
C) fluorescence
D) using an enzyme-linked reaction involving 4-chloro-1-naphthol

Question 68

Studying the entire complement of proteins being produced by a cell is called

A) genomics
B) proteomics
C) ELISA
D) western blot

Question 69

The purpose of using nitrocellulose for a western blot is to

A) allow the various proteins to migrate and separate
B) bind to the primary antibody
C) allow the proteins to be concentrated onto the face of the nitrocellulose where they will be accessible to the primary antibody
D) to allow the 4-chloro-1-naphthol to react and be visible

Question 70

Identify a proteolytic enzyme that preferentially hydrolyzes amide bonds adjacent to aromatic amino acid residues.

A) Lipase
B) Chymotrypsin
C) Amylase
D) Tyrosinase

Question 71

Which of the following is NOT true concerning a western blot?

A) it is usually performed in a 96 well microtiter plate
B) it involves the use of primary antibodies
C) it involves the use of secondary antibodies
D) it involves electrophoresis

Question 72

Which of the following polysaccharides is used to make up resins for use in electrophoresis and in column chromatography?

A) Fructan
B) Agarose
C) Callose
D) Glucan