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 . monoclonal antibody . epitope . antigen . immunofluorescence (direct and indirect) . chemiluminescence . conjugated double bonds . codon usage bias . promotor-reporter construct . sterical hindrance . overexpression artifact . bimodal fluorescence complementation (split YFP) . photoactivable FPs . CRAP . stromules . PALM/STORM
You know by what histochemical approaches you can detect amyloplasts, lignified cells, and lipid structures such as a Caspary Strip or wax layers
You can explain and apply the link between the chemical structure of a fluorochrome and its optical properties
You can explain, what indirect immunofluorescence is and why it is useful
You can explain the main two applications for GFP and what the requirements for the respective promoter are
You can critically reflect on artifacts linked with GFP live-cell imageing and suggest appropriate controls to handle them
You can explain why photoactivable FPs are so attractive and how they are used for super-resolution microscopy
Vertiefung (for Bachelor students)
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 right.
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.
- 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 his Nobel Prize Speech for this.
- 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?
- When free GFP is expressed, the nucleus appears green. Why?
- You are investigating a nucleus of a fibroblast by a confocal microscope equipped with an ArKr laser. Which of the following fluorochromes do you choose? DAPI . Phalloidin . Propidium Iodide . GFP . FIT
- You want to stain microtubules in chemically fixed moss leaflets. How do you generate the fluorescence? Using a secondar antibody conjugated to rhodamine, using a GFP fusion of tubulin, using a FITC-conjugated secondary antibody, or FITC-conjugated phalloidin?
Special topic (Master students)
Your task is to go through the desing of a GFP-based localisation study for a gene of interest. Details are given here and additional material provided on the Ilias page (slides, a recording, and a dataset you should analyse). The task is typical for molecular cell biology. Along with solving the task, there are a couple of questions given in the slides on this topic on Ilias, which you also should answer in your design.
- simple DNatube movie on the discovery of GFP and the significance of this discovery
- Youtube-movie on GFP as marker for neural stem cells
- Biography of Martin Chalfie with background, how he arrived to use GFP as reporter.