PARAMETERS WHICH AFFECT THE ESTIMATION OF PROTEIN MOBILITY
BY METHOD FRAP IN LIVING CELLS ON THE EXAMPLE OF PROTEIN FIBRILLARIN
V.V. Barygina,1,2 A.A. Mironova,1,3 O.V. Zatsepina 1
1 M M. Shemyakin—Yu. A. Ovchinnikov Institute of Bioorganic Chemistry RAS,
2 Faculties of Biology and 3 Bioengineering and Bioinformatics of M. V. Lomonosov Moscow State University;
e-mail: zatsepina_olga@mail.ru, v.barygina@mail.ru
Method FRAP (fluorescence recovery after photobleaching) in combination with confocal laser scanning
microscopy represents one of the principal approaches in studying the properties of proteins in living mammal
cells. However, the data of different authors on the dynamic properties of the same protein and even in cells of
the same type can differ greatly. The reasons of such discrepancies were not specifically analyzed yet. In the
present work, on the example of the nucleolar protein fibrillarin fused to EGFP, was studied the impact of area
of the region of interest (ROI) and temperature conditions on the main dynamic characteristics of the protein,
such as mobile fraction and time for half-time of fluorescence recovery after photobleaching (t1/2). Obtained results
suggest that both parameters have a great impact on the estimation of mobile properties of fibrillarin-EGFP
in HeLa cells. Was concluded that during FRAP experiments the area of ROI has to be standardized and, as possible,
minimized. Moreover, analyzing the dynamic properties of the nucleolar proteins, which take part in the
temperature-sensitive reactions, the standard temperature conditions should also be standardized.
Key words: FRAP, fibrillarin, mobile fraction, half-time of recovery (t1/2), area of bleaching, temperature conditions
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