Two questions in my homework are giving me trouble.
4. It is important to have controls in your experiment to rule out the possibility of an artifact (a false positive created by the experimental procedure). In our first experiment we amplified pEGFP-MannII DNA, which was transfected into HeLa cells, in order to visualize the Golgi Apparatus (MannII is a Golgi resident enzyme. It stays in the Golgi).What control are you missing in this experiment? If we add your control, what are the possible outcomes and respective implications? (2 points)
Two controls we DID have are a transfection with a known quantity of purified plasmid and a transfection with nothing. Hope that helps.
5. You do immunofluorescence by staining cells for calreticulin(stained in red, you saw this in Protocol #2, think about what it looked like) and protein A (a random protein I made up, stained in green). Looking in the microscope, it looks like these two proteins are co-localized, appearing to be in the same intracellular compartment (You see yellow. Remember that red light mixed with green light make yellow light). Based on this data, you say that calreticulin and protein A are in the same intracellular compartment but Sam does not agree with your conclusion. Why do you think he doesn’t agree? How would you definitively test whether these two proteins are in the same intracellular compartment or not, using a complimentary technique (something that is NOT immunofluorescence, since you already tried that and it’s not enough to convince Sam)? (2 points)
I know that Calreticulin is a resident ER protein. However, staining for the ER in a cell shows that it is evenly "smeared" across the entire cell except for a dark spot that is the nucleus.
