Confocal imaging and Frap

Edited by the Laboratorytalk editorial team Jul 29, 2003

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.