Molecular Biology Exam test quiz
and answers graded A+
Would it be possible to raise an antibody against another antibody? - ANS✅✅Yes. It is done by
introducing antibodies from one species into a second species, for example, by injecting mouse
antibodies from one species into a second species, for example, by injecting mouse antibodies into
goats.
- > mouse antibodies are recognized as foreign proteins by the goat to generate antibodies that bind
to the mouse antibodies.
- > can be done in the same species due to the idiotype of the antigen being recognized by the
antibody.
Purification technique in which a mixture of proteins is passed through a cylinder containing a pour
out solid matrix. - ANS✅✅Column chromatography
Artificial product generated by linking the coding sequences for two different proteins, or protein
segments, and expressing the hybrid gene in cells. - ANS✅✅Fusion protein
Technique where a protein mixture is separated by running it through a gel containing a detergent
that binds to and unfolds the proteins. - ANS✅✅SDS polyacrylamide-gel electrophoresis (SDS-
PAGE)
Technique where proteins are separated by electrophoresis, immobilized on a paper sheet, and then
analyzed usually by labeled antibodies. - ANS✅✅Western Blotting (immunoblotting)
Where is the anode (+) placed when running an electrophoresis gel? - ANS✅✅SDS detergent
carries a negative charge so that when it binds, the proteins become highly negative—> they will
move toward the positive anode, so you must place it at the bottom.
After developing an auto radiograph after a two-week exposure, you incubate your protein with cell-
cycle kinase in the presence of P32-ATP to demonstrate that it was a substrate for the kinase. *The
band on the gel is at the right position, but very faint.* You plan to put blot against film and expose
for a longer period of time, but advisor tells you to do it all aver again and use more radioactivity.
Why? - ANS✅✅The half-life of P32 is 14.3 days, so the film was already exposed for one half-life in
the first 2 weeks. You could expose the new film from now to the end of time and not have any
brighter bands.
, Western Blotting procedure - ANS✅✅1. Load and separate protein samples on SDS-PAGE.
2. Electrophoretically transfer fractionated proteins onto PVDF or nitrocellulose membrane.
3. Block the membrane with neutral protein (BSA or milk)
4. Incubate membrane with primary antibody specific to target protein. Wash.
5. Incubate the membrane with HRP-labeled secondary antibody specific to primary antibody. Wash.
6. Incubate the blot with chemiluminescent HRP substrate and expose to film.
Sensitivity of detection of immunoblotting. - ANS✅✅
You raised four different monoclonal antibodies to X-Orc1, which is a component of the DNA
replication origin recognition complex (ORC) found in eukaryotes. You want to use the antibodies to
immunopurify other members of ORC. To decide which of your monoclonal antibodies —TK1, TK15,
TK37, or TK47- is best suited for this purpose, you covalently attach them to beads, incubate them
with X egg extract, spin the beads down, and wash them, and then solubility the bound proteins with
SDS. You use the SDS-PAGE to separate the solubilized proteins and stain them.
A. How do you interpret bands that arise from the proteins present in ORC?
B. Why does the various monoclonal antibodies give such different results?
C. How would you know which antibody is the best to use?
D. How would you determine which band on the gel is the target? - ANS✅✅A. All four monoclonal
antibodies were raised against Orc1-> all should bind to Orc1 at minimum. (Band in every lane)
- also bind to all other members of class of protein.
B. Each monoclonal antibody is presumably specific for a particular epitope on Orc1. The lane with
the fewest bands suggests that it's target epitope is only on Orc1.
C. You would want to use the most specific antibody, which has the fewest amount of bands on the
blot.
D. You could detect Orc1 by W.B using the most specific antibody as the primary.
Why would a solution of imidazole release a histidine-tagged protein from an Ni2+ column. -
ANS✅✅Imidazole is the ring of histidine.—> competes with His for binding to the Ni2+ column—>
releasing the protein.
Large protein complex in the cytoskeleton and nucleus with proteolytic activity that is responsible
for degrading proteins marked for destruction. - ANS✅✅Proteasome
and answers graded A+
Would it be possible to raise an antibody against another antibody? - ANS✅✅Yes. It is done by
introducing antibodies from one species into a second species, for example, by injecting mouse
antibodies from one species into a second species, for example, by injecting mouse antibodies into
goats.
- > mouse antibodies are recognized as foreign proteins by the goat to generate antibodies that bind
to the mouse antibodies.
- > can be done in the same species due to the idiotype of the antigen being recognized by the
antibody.
Purification technique in which a mixture of proteins is passed through a cylinder containing a pour
out solid matrix. - ANS✅✅Column chromatography
Artificial product generated by linking the coding sequences for two different proteins, or protein
segments, and expressing the hybrid gene in cells. - ANS✅✅Fusion protein
Technique where a protein mixture is separated by running it through a gel containing a detergent
that binds to and unfolds the proteins. - ANS✅✅SDS polyacrylamide-gel electrophoresis (SDS-
PAGE)
Technique where proteins are separated by electrophoresis, immobilized on a paper sheet, and then
analyzed usually by labeled antibodies. - ANS✅✅Western Blotting (immunoblotting)
Where is the anode (+) placed when running an electrophoresis gel? - ANS✅✅SDS detergent
carries a negative charge so that when it binds, the proteins become highly negative—> they will
move toward the positive anode, so you must place it at the bottom.
After developing an auto radiograph after a two-week exposure, you incubate your protein with cell-
cycle kinase in the presence of P32-ATP to demonstrate that it was a substrate for the kinase. *The
band on the gel is at the right position, but very faint.* You plan to put blot against film and expose
for a longer period of time, but advisor tells you to do it all aver again and use more radioactivity.
Why? - ANS✅✅The half-life of P32 is 14.3 days, so the film was already exposed for one half-life in
the first 2 weeks. You could expose the new film from now to the end of time and not have any
brighter bands.
, Western Blotting procedure - ANS✅✅1. Load and separate protein samples on SDS-PAGE.
2. Electrophoretically transfer fractionated proteins onto PVDF or nitrocellulose membrane.
3. Block the membrane with neutral protein (BSA or milk)
4. Incubate membrane with primary antibody specific to target protein. Wash.
5. Incubate the membrane with HRP-labeled secondary antibody specific to primary antibody. Wash.
6. Incubate the blot with chemiluminescent HRP substrate and expose to film.
Sensitivity of detection of immunoblotting. - ANS✅✅
You raised four different monoclonal antibodies to X-Orc1, which is a component of the DNA
replication origin recognition complex (ORC) found in eukaryotes. You want to use the antibodies to
immunopurify other members of ORC. To decide which of your monoclonal antibodies —TK1, TK15,
TK37, or TK47- is best suited for this purpose, you covalently attach them to beads, incubate them
with X egg extract, spin the beads down, and wash them, and then solubility the bound proteins with
SDS. You use the SDS-PAGE to separate the solubilized proteins and stain them.
A. How do you interpret bands that arise from the proteins present in ORC?
B. Why does the various monoclonal antibodies give such different results?
C. How would you know which antibody is the best to use?
D. How would you determine which band on the gel is the target? - ANS✅✅A. All four monoclonal
antibodies were raised against Orc1-> all should bind to Orc1 at minimum. (Band in every lane)
- also bind to all other members of class of protein.
B. Each monoclonal antibody is presumably specific for a particular epitope on Orc1. The lane with
the fewest bands suggests that it's target epitope is only on Orc1.
C. You would want to use the most specific antibody, which has the fewest amount of bands on the
blot.
D. You could detect Orc1 by W.B using the most specific antibody as the primary.
Why would a solution of imidazole release a histidine-tagged protein from an Ni2+ column. -
ANS✅✅Imidazole is the ring of histidine.—> competes with His for binding to the Ni2+ column—>
releasing the protein.
Large protein complex in the cytoskeleton and nucleus with proteolytic activity that is responsible
for degrading proteins marked for destruction. - ANS✅✅Proteasome
