Biotechnology Chapter 9 Day 1 Questions
Note: As usual, some of the questions go beyond the information that the book has provided. Google is your friend.
- When performing a two-dimensional PAGE experiment, why would you use a zwitterionic detergent during the first-dimensional separation and a negatively-charged detergent, such as SDS, for the second dimension?
- Some proteins, such as IRF3, can be phosphorylated many times. Would you expect a phosphate group to alter a protein’s isoelectric point or size? If so, would the spots from the variously phosphorylated forms of IRF3 be more noticeable in the x- or y-axis of the 2D gel?
- Compare and contrast microarray raw data (figure 8.19) with two-color 2D gel raw data.
- Why might a scientist cut out a “spot” from a 2D gel?
- One problem with 2D-DIGE is that the dynamic range is narrow. Explain what this means and give examples of proteins that exceed the range limits.
- The book states, “Antibodies are extremely specific and will bind only to one target protein.” This isn’t always true. Describe why cross-reactivity could be detrimental in Western blot experiments but beneficial in an immune response using vaccinia virus as an example.
- Describe why proteins will stick to nitrocellulose paper. Why is “blocking” an important step of the Western blot? Why does the blocking step precede the addition of primary antibody?
- We discussed primary and secondary antibodies for ELISAs. Discuss the roles of primary and secondary antibodies in a Western blot. Draw a picture to help explain your discussion of these two types of antibodies. Which antibody (primary or secondary) would have an enzyme conjugated to it?
- In both DNA and protein gel electrophoresis, we see a pattern where the smaller macromolecules move through the gel faster than the larger macromolecules. Why is the pattern reversed in a size-exclusion chromatographic column?
- Compare and contrast reverse-phase HPLC and ion-exchange chromatography. How do you elute the strongest “binders” in each of these types of chromatography?
- If you are eluting proteins off of an HPLC column, what wavelength of UV light would be appropriate to monitor?
- Describe how proteases are categorized.