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This lecture covers the use of fluorescently tagged proteins in research
Fluorescent proteins allow us to visualise proteins in living cells in real time
The effects of external factors such as hormones, growth factors or drugs can be studies on the labelled protein
Fluorescent resonance energy transfer enables the direct interaction between two proteins to be studied within a cell
Learning objectives:
By the end of your revision, you should be able to explain what fluorescent tagged proteins are. You should understand the different situations in which they are useful and be able to design an experiment in which they are used. You should also appreciate the potential of fluorescence resonance energy transfer and possible pitfalls in its use.
Background reading:
The green fluorescent protein
Tsien RY
Annual Review of Biochemistry (1998) 67:509-544.
Studying nuclear receptors with green fluorescence
Hager GL,
Methods in Enzymology (1999) 302:73-86.
Fluorescence resonance energy transfer microscopy of localised protein interactions in the living cell nucleus
Day RN, Periasamy A, Schaufele F,
Methods (2001) 25:4-18.
Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and proliferator activated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are required for transcription
Llopis J et al.,
Proc. Natl. Acad. Sci. USA (2000) 97: 4363-4368.
nuclear hormone receptors can be imaged in live cells and are required for transcription
Fluorescent proteins
Green Fluorescent Protein (GFP)
characteristics
Isolated from the bioluminescent jellyfish Aequoria victoria
A fluorescent protein which emits blue light with a peak near 470 nm.
Structure:
Size 27 kDa
11 stranded beta barrel with
Central helix passing through the centre of the barrel
Contains fluorophore
Independent
Stabile
Fluorophore
tripeptide Ser65-Tyr66-Gly67
Imidazolinone ring structure
Absorbs: major excitation peak at 395 nm
minor peak
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