Your absent-minded colleague is studying a new protein called Mario, and they ha

Question

Your absent-minded colleague is studying a new protein called Mario, and they have made three cell lines: one contains a wild-type (no mutation) copy of Mario, one has a deletion mutation in Mario, and one contains a plasmid that overexpresses Mario. Unfortunately, your colleague has mixed up which cell line is which. Even worse, they've forgotten the size of the protein! To clear things up, your colleague creates a cell homogenate from each cell line and runs them in TE Lanes A-C of an SDS-PAGE gel shown below. Lanes MW A B C 50KD Figure adapted from Sivashanmugam, A et al., Protein Sci., 2009 18(5): 936-948. 37kD A. What feature of the gel provides information about the expression levels of proteins? 25kD B. Which of these Lanes corresponds to: 1. The wild-type cell line? C 2. The cell line with a deletion in Mario? A 3. The cell line with a plasmid to overexpress Mario?6 se nis cell ine more yheon the oher tu the Cek line· s: dianinishin compared&Mano;'s Please explain your reasoning: cell lne z) A because ceul exp s B because the cel lice is more ohincel ahic the overexpressed cell op Mano C. What feature of the gel provides information about the size of proteins? What do you think the size of Mario is? D. Y organelle as Mario. How could you test this hypothesis using cell fractionation by centrifugation and SDS-PAGE? ou have identified another protein, Luigi, that you predict is located in the same

Explanation / Answer

WT = Normal level of protein
Deletion mutant = Less/No protein
Overexpression = More protein

A.
Variation in the relative intensity of a particular band provides information about the nature of the sample.

B.
WT = Normal level of protein = C
Deletion mutant = Less/No protein = A
Over expression = More protein = B

C.
Using MW marker enables us to find out the approximate MW of an unknown protein.
Mario MW is approximately 35 kDa

D.
Perform cell fractionation to separate each organelle.
Perform SDS-PAGE for the fraction in which Mario is present.
Use an antibody specific to Luigi to probe its presence.
If Luigi is present in the same organelle as that of Mario, we can detect a band in the Western blotting. Otherwise, No band can be observed.

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