Evaluation of the Dynamicity of Mitotic Exit Network and Spindle Position Checkpoint Components on Spindle Pole Bodies by Fluorescence Recovery After Photobleaching (FRAP).
Fluorescence recovery after photobleaching (FRAP) is a powerful technique to study in vivo binding and diffusion dynamics of fluorescently labeled proteins. In this chapter, we describe how to determine spindle pole body (SPB) binding dynamics of mitotic exit network (MEN) and spindle position checkpoint (SPOC) proteins using FRAP microscopy. Procedures presented here include the growth of the yeast cultures, sample preparation, image acquisition and analysis.
DKFZ-ZMBH Alliance, German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 280, Heidelberg, 69120, Germany.|Centre for Organismal Studies (COS), University of Heidelberg, Im Neuenheimer Feld 230, Heidelberg, 69120, Germany.|Department of Molecular Biology and Genetics, Gebze Technical University, Gebze/Kocaeli, 41400, Turkey.
Ayse Koca
AK
Caydasi
DKFZ-ZMBH Alliance, German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 280, Heidelberg, 69120, Germany. gislene.pereira@cos.uni-heidelberg.de.|Centre for Organismal Studies (COS), University of Heidelberg, Im Neuenheimer Feld 230, Heidelberg, 69120, Germany. gislene.pereira@cos.uni-heidelberg.de.
Gislene
G
Pereira
D018797:Cell Cycle Proteins / Q000032:analysis* / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D059566:M Phase Cell Cycle Checkpoints; D008856:Microscopy, Fluorescence / Q000379:methods*; D008938:Mitosis; D061848:Optical Imaging / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000378:metabolism; D064048:Spindle Pole Bodies / Q000378:metabolism* / Q000648:ultrastructure
D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins
Binding dynamics; Centrosome; FRAP; MEN; SPB; SPOC; Spindle pole body; Spindle position checkpoint