Question

You are using budding yeast (Saccharomyces cerevisiae) as a model system to study the function of uncharacterized proteins. You identify a new protein, Pro3500, that you predict plays a role in mRNA processing, based on its similarity to other mRNA processing factors. You take advantage of the many genetic and molecular tools available in budding yeast to perform a series of experiments investigating the function of Pro3500.

Based on its predicted role in mRNA processing, you expect Pro3500 to localize to the nucleus, however you need to determine this experimentally. Which of the following techniques would be the best, most direct method to determine the localization of Pro3500?

1. SDS-PAGE and western blotting using an antibody to Pro3500
2. fluorescence microscopy using an antibody to Pro3500
3. performing a genetic screen to identify temperature sensitive mutations in Pro3500
4. chromatography followed by mass spectrometry

flow cytometry

Answer 1

1. Answer: Option B is correct
Explanation:
To study the localization of a protein, we can translationally fuse the protein with a fluorescent marker such as GFP or RFP. The fluorescence signal can be detected in the nucleus if the protein is localized to the nucleus.

We can also use a fluorescent antibody against the respective protein to study its localization in the cell.

SDS-PAGE followed by Western blotting is used to identify the quality and quantity of a protein sample.
Temperature-sensitive mutants exhibit a mutant phenotype at the restrictive temperature.
Chromatography followed by mass spectrometry is used to separate proteins based on their size.