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Vorbereitung

Web tutorial of  Vienna University on fluorescence

simple DNatube movie on the  discovery of GFP and the significance of this discovery

Nachbereitung

General on GFP

 

VERTIEFUNG (Master)

Localisation study

What we expect you to know

Terminology

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 . 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

Molecular Probes - 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. You want to test, in which cells of the model plant Arabidopsis thaliana the receptor-like kinase too-many mouths (tmm) is expressed.

Which of the following constructs would be suited (explain for each construct, why or why not you would select it for this purpose).

  • 35S::tmm-GFP (C-terminal fusion)
  • 35S::GFP-tmm (N-terminal fusion)
  • tmm::GFP
  • tmm::tmm-GFP (C-terminal fusion)
  • tmm::GFP-tmm (N-terminal fusion)
terminology: the label before :: describe the promoter, fusions on the protein level are indicate by -
 

4. When free GFP is expressed, the nucleus appears green. Why?