Question 1

Explain the molecular basis of an RNase protection assay. How can this method be used to map the transcription start site for a gene?

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Question 2

What kinds of information can be obtained from a Western blot? Diagram the key steps in the method.

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Question 3

Explain the molecular basis for protein separation by isoelectric focusing (the first step of 2D-PAGE).

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Question 4

You have used a polyclonal primary antibody made in rabbit to detect your protein of interest on Western blot. Give an example of an appropriate secondary antibody to use for detection. Explain your choice.

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Question 5

Compare and contrast the steps in production of polyclonal and monoclonal antibodies.

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Question 6

Diagram the key steps in a "sandwich" ELISA. What information can be obtained from this type of assay?

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Question 7

Compare and contrast the electrophoretic mobility shift assay (EMSA) and DNase I footprinting methods for assaying specific DNA-protein interactions. What information does DNase footprinting provide that EMSA does not?

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Question 8

You want to confirm the interaction of a particular protein with a specific sequence of DNA within the context of a living cell. What technique would you use and why?

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Question 9

You want to confirm the interaction of a particular protein with another protein at a precise location within a cell. What technique would you use and why?

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Question 10

Diagram the key steps in a GST pull-down assay. Is the GST-tagged protein the "bait" or the "prey"?

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Question 11

In a yeast two-hybrid assay that uses lacZ as a reporter gene, what does the production of a blue product signify?

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Question 12

Compare and contrast the type of information that can be obtained from X-ray crystallography with nuclear magnetic resonance (NMR) spectroscopy.

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Question 13

Compare and contrast the type of information that can be obtained from cryoelectron microscopy with atomic force microscopy.

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Question 14

You have purified a protein. When you subject it to SDS-PAGE, two bands are seen. Provide a possible explanation and describe how you could test your hypothesis.

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Question 15

Consider a Northern blot:
(a) Which strand of a DNA probe will hybridize to mRNA on the blot: the anti-sense strand, the sense strand, or both strands?
(b) On the diagram of the DNA-RNA hybrid shown below, which bonds form when the DNA probe hybridizes to the mRNA?

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(a) Which strand of...

Question 16

Consider a cotransfection assay with expression vectors for two putative transcription factors that you suspect work together to activate transcription, and with a regulatory region of interest attached to a CAT reporter gene. You also test two deletion () mutants of the regulatory region. 
(a) How could you confirm that the transcription factors are transcribed and translated in the transfected cells?
(b) Interpret the results of the CAT assay below. The plus and minus symbols indicate that presence or absence of expression vectors for the listed components in the transfection assay.
  (c) How would you demonstrate protein-protein interaction between the two transcription factors in vivo and in vitro? Show sample positive results.
(d) Describe protocols to further characterize direct binding of the proteins to DNA in vitro and in vivo. Show sample results.

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Question 17

You have transfected siRNA against transcription factor A from Q. 59 into cells that normally express the transcription factor.
(a) What would be the effect on CAT reporter gene expression?
(b) How could you determine whether RNAi acts at the level of mRNA or translation in this case?

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Quiz 9: Tools for Analyzing Gene Expression