What we expect you to know
You should know, what these terms mean and you should be able to explain them in your own words.
Histochemistry . DAPI . FITC . Rhodamin . BODIPY dye . Alexa dye . FM4-64 . Höchst . ROS . Rhodamin G6 Cl . Mitotracker . ER-Tracker . DiOC . Propidiumiodide . phalloidin . monoclonal antibody . epitope . antigen . immunofluorescence (direct and indirect) . aequorin . chemiluminescence . coelenterazine . GFP . conjugated double bonds . codon usage bias . promotor-reporter construct . fusion protein . native promotor . constitutive promotor . sterical hindrance . overexpression artifact . bimodal fluorescence complementation (split YFP) . timer GFP . DsRed . Stokes shift . mEOS . photoactivable FPs . CRAP . stromules . PALM/STORM
Fluorescent Markers - Exercise
1. You have cloned a new microtubule-associated protein from rice and you want to test, to which microtubules it can bind. You have purified the protein from rice extracts and you have now obtained antibodies after injecting the purified protein into rabbits. To stain microtubules, you use a specific monoclonal anti-tubulin antibody from mouse. To see the colocalisation, you use anti-mouse IgG FITC and anti-rabbit IgG Rhodamin. You observe in dividing cells the image shown at the left.
The very close association of your protein with microtubules makes you suspicious - it is much stricter than you expected. You want to test two points:
A. is there cross-reactivity between your anti-MAP antibody and tubulin?
B. is there filter leakage (i.e. your optical set-up allows the FITC Signal from microtubules to leak through the Rhodamin filter)
Propose two negative controls for this experiment.
2. For what application did Martin Chalfie plant to use GFP? What alternative approaches existed at that time? Why was he initially sceptical, whether the GFP approach would work. You have to read for this his Nobel Prize Speech.
3. Transgenic BY-2 cells often loose their fluorescence after some time. To avoid this, one needs to plate them after selection once more on low density and then pick clonal strains, which are then stable over years. Do you have an idea, why this is not the case, if you just go on to cultivate the original transformed cell culture?
4. When free GFP is expressed, the nucleus appears green. Why?