What should the fluorescence pattern of that compartment look like with the fluorescent protein?
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g., nucleus, mitochondria, ER, Golgi, plasma membrane, lysosome, cytoskeleton, etc.). Show more…
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Scientists want to design fluorescent marker proteins for different cellular compartments using the Green Fluorescent Protein (GFP). This means that the GFP will be targeted and glow at the organelle of interest and they can use these to mark different organelles during fluorescence microscopy. To make these marker proteins, they take the sequence of GFP and add the desired sorting signal. A. GFP on its own is an excellent protein to use as an organelle marker protein because it is fluorescent. That means once a sorting signal is added it will light up in the indicated organelle. But there is an additional reason why GFP is well suited for this job. It doesn't have any additional sorting signals. Where might you expect to find GFP with nothing else added? Explain your answer. (2 mark) B. On the domain maps provided below, indicate the location and type (ER import, NLS, M6P) of all targeting sequences that you predict would have to be added for GFP to end up in the location given. Note: some sequences do not have a location requirement. These can be put in any portion of the protein. If you don't need to add any targeting signals, clearly indicate this in your answer – a blank will NOT be considered as an answer for marks. (2 marks each): 1. GFP as a soluble ER resident marker protein 2. GFP as a mitochondrial matrix marker protein 3. GFP as a nuclear marker protein 4. GFP as a plasma membrane marker protein, with 1 transmembrane domain, and its N-terminus on the outside of the cell.
Adi S.
The green fluorescent protein (GFP) is normally a cytosolic protein. You have genetically engineered a form of GFP that contains a KDEL sequence. When the construct containing this new protein (GFP with KDEL) is transfected into normal human fibroblasts and examined using fluorescence microscopy, the fluorescence appears throughout the cytoplasm, as shown in the diagram on the right. A. How would you explain this pattern given that KDEL is supposed to be an ER-specific sorting sequence? To analyze the results further, fractions of different organelles and the cytoplasm were collected from cells expressing this KDEL-containing GFP construct and then examined on Western blots using antibodies against GFP (27 kDa) and protein disulfide isomerase (PDI), a protein that is normally found in the rough ER (RER) and is approximately 55 kDa. The blot confirms the presence of GFP exclusively in the cytoplasm, and as expected, a PDI signal in the RER fraction. B. How would you explain the PDI band, albeit weak, in the Golgi fraction? C. Given the function of PDI proteins, what would you expect if both alleles of a PDI gene were knocked out in mice? [Think about what they do and how essential they are, if at all] The antibodies used above do not detect any signals in the nuclear fraction, which indicates their specificity or the fact that no proteins were isolated and loaded in the nuclear fraction lane. D. What antibody could be used to show there were nuclear proteins present in this sample? [Just think of an appropriate protein that you could make an antibody against. There are many correct answers]. Justify your choice.
Madhur L.
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.
Josee P.
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