Chapter 5 Techniques in Protein Biochemistry
Matching Questions
Use the following to answer questions 1-10:
Choose the correct answer from the list below. Not all of the answers will be used.
a) HPLC
b) specific activity
c) MALDI-TOF mass spectrum
d) gradient centrifugation
e) proteome
f) SDS
g) two-dimensional electrophoresis
h) Svedberg
i) immunoglobulin
j) differential centrifugation
k) overlap peptides
l) affinity chromatography
1.
The ratio of enzyme activity relative to total protein is called ____________.
2.
The first step in protein purification from a homogenate is usually ____________.
Ans:
Section: 5.2
3.
____________ A type of purification that is based on the attraction of the protein for a particular
chemical group.
Ans:
Section: 5.2
4.
____________ can be added prior to gel electrophoresis to denature the proteins.
Ans:
5.
Sedimentation coefficients are expressed in ____________ units.
Ans:
h
Section: 5.3
Ans:
b
Section: 5.2
Chapter 5 Techniques in Protein Biochemistry
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6.
Proteins with different sedimentation coefficients can be separated by ____________.
7.
In order to sequence a whole protein, ____________ are used.
Ans:
k
Section: 5.4
8.
____________ The subset of gene products actually expressed by the cell.
Ans:
Section: 5.1
9.
____________ A protein purification technique characterized by high resolution and rapid
separation.
Ans:
Section: 5.2
10.
____________ The separation of proteins based on charge then size.
Ans:
g
Section: 5.2
Fill–in-the Blank Questions
11.
Proteins can be separated from small molecules and ions through a semi-permeable membrane
by .
Ans: dialysis Section: 5.2
12.
Molecular exclusion gel or gel-filtration chromatography separates molecules on the basis of .
Ans: size Section: 5.2
13.
In amino acid composition analysis, amino acids are visualized after separation using the
chemical reagent .
Ans: fluorescamine Section: 5.4
14.
In the Edman procedure for peptide sequence, phenyl isothiocyanate is used to selectively
remove the residue as a PTH-derivative.
Ans: amino-terminal Section: 5.4
15.
Antibodies used as reagents to quantify proteins or other antigens is the basis for the technique
called .
Ans: ELISA (enzyme-linked immunosorbent assay) Section: 5.3
Ans:
d
Section: 5.3
Chapter 5 Techniques in Protein Biochemistry
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16.
allows the detection of small amounts and the size of target proteins.
17.
Polypeptides can be fragmented into smaller peptides by cleavage with chymotrypsin, which
hydrolyzes the peptide bond at the C-terminal side of residues.
Ans: phenyalanine, tyrosine, and tryptophan Section: 5.4
18.
gels are often used as the media for electrophoretic techniques such as SDS-PAGE and
isoelectric focusing.
Ans: Polyacrylamide Section: 5.4
19.
The of a protein is the pH at which its net charge is zero.
Ans: isoelectric point Section: 5.2
Multiple-Choice Questions
20.
When enzymes are purified, the assay is often based on:
A)
light absorbance.
D)
B)
catalytic activity.
E)
C)
pH.
Ans: B Section: 5.2
21.
Receptor proteins are often assayed using:
A)
binding assays.
D)
B)
enzymatic activity.
E)
C)
amino acid analysis.
Ans: A Section: 5.3
22.
What is the advantage of adding SDS to gel electrophoresis?
A)
SDS colors the proteins for visualization.
B)
SDS reduces disulfide bonds.
C)
SDS allows proteins to be separated on the basis of approximate mass.
D)
None of the above.
E)
All of the above.
Ans: C Section: 5.2
23.
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
Ans: D Section: 5.2
Ans: Western blotting or immunoblotting Section: 5.3
Chapter 5 Techniques in Protein Biochemistry
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24.
Which of the following affects the sedimentation of a particle?
A)
mass
D)
B)
shape
E)
C)
the density of the solution
25.
Cyanogen bromide cleaves the peptide bond at:
A)
the carboxyl side of Arg and Lys residues.
B)
the carboxyl side of Met residues.
C)
the amino terminus.
D)
None of the above.
E)
All of the above.
Ans: B Section: 5.4
26.
Trypsin cleaves the peptide bond at:
A)
the carboxyl side of Arg and Lys residues.
B)
the carboxyl side of Met residues.
C)
the amino terminus.
D)
None of the above.
E)
All of the above.
Ans: A Section: 5.4
27.
Which of the following techniques can be used to determine the size of a target protein?
A)
Edman degradation
D)
B)
affinity chromatography
E)
C)
western blot
Ans: C Section: 5.3
28.
What types of molecules can serve as antigens?
A)
proteins
D)
B)
polysaccharides
E)
C)
metal ions
Ans: E Section: 5.3
29.
Affinity chromatographs:
A)
allow high resolution and rapid
separation.
D)
B)
separate proteins based on size.
E)
C)
separate proteins based on charge.
Ans: D Section: 5.2
30.
What conditions could cause changes in the proteome of a cell?
A)
developmental stage
B)
environmental condition
C)
enzymatic modification
D)
All of the above.
E)
None of the above.
Ans: D Section: 5.1
Ans: D Section: 5.3
Chapter 5 Techniques in Protein Biochemistry
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31.
Which technique cannot be used for quantitative analysis?
A)
gradient centrifugation
D)
B)
ELISA
E)
C)
enzyme assay
32.
Which of the following is true regarding gel filtration chromatography and PAGE?
A)
In both, small proteins move most
rapidly.
D)
B)
In both, large proteins move most
rapidly.
E)
C)
In PAGE, large proteins move most
rapidly but in gel filtration, small
proteins move most rapidly.
Ans: D Section: 5.2
33.
Two proteins are similar in size but differ significantly in the number of acidic and basic amino
acids. Which of the following techniques would be best suited to 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)
None of the above.
Ans: D Section: 5.2
34.
Two proteins are similar in the number of acidic and basic amino acids but are different
significantly in size. Which of the following techniques would be best suited to 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)
None of the above.
Ans: A Section: 5.2
35.
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)
None of the above.
Ans: C Section: 5.2
Ans: A Section: Entire Chapter
Chapter 5 Techniques in Protein Biochemistry
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36.
You have isolated a protein, but by the time you have gotten it pure, you have only enough
sample to do one type of analysis. Which of the following would you choose and why?
A)
MALDI-TOF mass spectrometry to determine as much sequence data as you can.
B)
ELIZA to identify any antigenic determinants.
C)
Amino Acid Composition Analysis because it can be done for the whole protein.
D)
2D gel electrophoresis to determine charge and size data of the protein.
E)
Salting out to concentrate the protein for further study.
37.
You are interested in studying a powerful enzyme that is expressed in low amounts. Which of
the following would you choose to determine how much is found in the tissue of interest?
A)
MALDI-TOF mass spectrometry to determine as much sequence data as you can.
B)
ELIZA to identify any antigenic determinants.
C)
Amino Acid Composition Analysis because it can be done for the whole protein.
D)
2D gel electrophoresis to determine charge and size data of the protein.
E)
Salting out to concentrate the protein for further study.
Ans: B Section: 5.4
Short-Answer Questions
38.
Why is an assay necessary for protein purification studies?
protein from the other cellular material.
Section: 5.2
39.
How is lactic acid dehydrogenase assayed?
nm, and the reaction can be monitored by the increase in absorbance at this wavelength.
Section: 5.2
40.
How do gel-filtration and ion-exchange chromatography differ?
the protein’s charge and affinity for the column media.
Section: 5.2
41.
How can a protein’s isoelectric point be used in protein purification?
Section: 5.2
Ans: A Section: 5.4
Chapter 5 Techniques in Protein Biochemistry
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42.
What is the purpose of determining the specific activity, yield, and purification level of a protein
purification protocol?
43.
What type of information can be obtained from gradient centrifugation?
Ans:
This technique can be used to determine mass and density, and to investigate molecular
shape and molecular interactions.
Section: 5.3
44.
Describe the Edman degradation method for protein-sequence analysis.
be identified by chromatography. The steps are repeated to identify the next amino acid
exposed at the amino terminus.
Section: 5.4
45.
How can the amino acid sequences be used to design a DNA probe?
Ans:
Using the amino acid sequence, the genetic code and a process known as reverse genetics,
a DNA sequence can be designed. (Codon degeneracy must be considered in the design.)
Section: 5.4
46.
Explain the process of immunoprecipitation.
the protein to be purified binds. The unbound proteins are washed away leaving only the
purified protein attached to the antibody.
Section: 5.4
47.
Explain the trend of specific activity as a protein is purified.
higher ratio of units of enzyme to total protein. Thus, as a protein is purified the specific
activity is increased
Section: 5.2
48.
List five possible steps in protein purification. Start with a technique used on a complex mixture
of proteins such as a cell lysate through a series of steps to a pure protein.
Ans:
homogenization, salting out, ion-exchange chromatography, gel filtration
chromatography, affinity chromatography
Section: 5.2
49.
Why is there a need for different digestion tools when fingerprinting a protein?
Ans:
To generate overlapping peptides, short pieces of peptides are generated with one or two
proteases and then further digested with other proteases.
Section: 5.4
selectively isolating the protein while maintaining its presence and activity. In order to
successfully purify protein, both the yield and purification level must remain high.
Section: 5.2
Chapter 5 Techniques in Protein Biochemistry
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50.
A protein that on a SDS PAGE runs as a single 25,000 dalton band runs as a 75,000 dalton band
on a native gel. Why?