The use of fluorescence recovery after photobleaching (Frap) to study the movement of a nucleolar protein in HeLa cells has been described in an application note
The use of fluorescence recovery after photobleaching (Frap) to study the movement of a nucleolar protein in HeLa cells has been described in an application note published by Bio-Rad.
Several recent studies show that some cellular proteins are rapidly and constantly moving between compartments within the cell.
Nobuaki Kikyo and his colleagues at the University of Minnesota used a Bio-Rad Radiance2100 confocal system to better understand the kinetics of association and disassociation of individual proteins in the nucleolus.
The application note describes the dynamic disassembly and re-assembly of the nucleolus in the context of somatic nuclear cloning and demonstrates the movement of GFP-tagged nucleolar protein B23 using the Frap technique.
Frap is a non-invasive technique with numerous applications in cell biology.
The technique enables observation of dynamic movement of fluorescence-tagged molecules within living cells under light microscopes.
Advances in imaging technology and the cloning of green fluorescence protein (GFP) have contributed significantly to Frap's growing popularity as a technique in cell biology. Frap introduces fluorescence-tagged molecules into living cells, usually by transfection of GFP-tagged protein into cells.
If a small area of the cell is photobleached and the fluorescence intensity recovers, it indicates influx of the fluorescence-tagged molecule from the surrounding non-bleached area of the cell.
This is evidence that the tagged protein is mobile within the cell.
Dr Kikyo describes fluorescent measurements performed with a Bio-Rad Radiance2100 confocal imaging system in conjunction with a Zeiss Axioskop2 microscope. The Radiance2100 system has a multi-phase time course option that allows for FRAP-style experiments to be performed.
The full details and results of this study may be read in Bio-Rad's application note 37, which is available free of charge by going to the Cell Science division on Bio-Rad's web site.
