Q1. When a protein of interest is tagged with a fluorescent protein-like GFP, what feature of this fusion protein must be considered and tested before interpreting any experimental results? If this feature is not considered, explain why the researchers will have difficulty interpreting their results. Q2. Compound light microscopes and transmission electron microscopes produce an image based on light transmission through the sample. Explain how the fluorescence microscope layout differs from that of transmission microscopes. Include the source of the light visualised in fluorescence microscopy in your answer.
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This includes factors such as the stability of the fluorescence, the quantum yield of the fluorescence, and how mutations or modifications to the GFP tag can affect its fluorescent properties. Show more…
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When a protein of interest is tagged with a fluorescent protein like GFP, what feature of this fusion protein must be considered and tested before interpreting any experimental results? If this feature is not considered, explain why the researchers will have difficulty interpreting their results.
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The protein-synthesizing machinery was isolated from eukaryotic cells and briefly treated with a low concentration of RNase. The sample was then subjected to sucrose gradient centrifugation. The gradient was fractionated and the absorbance, or optical density (OD), at $254 \mathrm{nm}$ was recorded for each fraction. The following plot was obtained. (a) What do the three peaks of absorbance in graph $A$ represent? The experiment was repeated except that, this time, the RNase treatment was omitted. (b) Why is the centrifugation pattern in graph B more complex? What do the series of peaks near the bottom of the centrifuge tube represent? Before the isolation of the protein-synthesizing machinery, the cells were grown in low concentrations of oxygen (hypoxic conditions). Again the experiment was repeated without RNase treatment (graph $\mathrm{C}$ ). (c) What is the effect of growing cells under hypoxic conditions? (GRAPH CAN'T COPY)
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