Protein analysis (part 1)

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.

1. 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?
2. 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?
3. Compare and contrast microarray raw data (figure 8.19) with two-color 2D gel raw data.
4. Why might a scientist cut out a “spot” from a 2D gel?
5. 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.
6. 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.
7. 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?
8. 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?
9. 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?
10. Compare and contrast reverse-phase HPLC and ion-exchange chromatography. How do you elute the strongest “binders” in each of these types of chromatography?
11. If you are eluting proteins off of an HPLC column, what wavelength of UV light would be appropriate to monitor?
12. Describe how proteases are categorized.