Biology Reference
In-Depth Information
Important considerations are discussed for designing experiments, collecting data,
and performing analysis. N&B analysis provides a robust method for assessing mem-
brane binding and assembly properties of peripheral proteins and lipid-binding
modules.
INTRODUCTION
Fluctuation analysis has been used to monitor protein oligomerization (
Barnwal,
Devi, Agarwal, Sharma, & Chary, 2011; Wang, Graveland-Bikker, de Kruif, &
Robillard, 2004
) as it serves as the basis for dynamic light scattering and fluores-
cence correlation spectroscopy (FCS). While dynamic light scattering requires many
molecules to produce a signal, FCS can detect single molecules in complex mixtures
such as live cells or solutions containing lipids and proteins. FCS was developed
in the 1970s (
Magde, Elson, &Webb, 1972, 1974
) and can detect fluctuation changes
in fluorescence while characterizing molecular brightness of the particles. FCS has
become more applicable to biologists and biochemists as its use was applied to live
cell studies (
Berland, So, & Gratton, 1995; Schwille, Bieschke, & Oehlenschl¨ger,
1997; Schwille, Korlach, & Webb, 1999
), and more recently, scanning confocal
microscopes have been used (
Digman, Brown, et al., 2005; Digman, Sengupta,
et al., 2005
). Fluorescence intensity fluctuations are often due to protein conforma-
tion changes, protein binding to immobile or less mobile fractions, or the entry and
exit of molecules through the area being imaged. In FCS, a fixed observation volume
is used to detect fluctuations in fluorescence intensity as single molecules pass
through the detection area. However, FCS alone does not account for the location
of fluorescent particles as a function of time so a spatial correlation approach can be
used in order to associate changes in fluorescent amplitude at one spot with changes
in nearby positions. A change in the correlation function of FCS can be performed
with raster image correlation spectroscopy (RICS) (
Digman & Gratton, 2009;
Digman, Stakic, & Gratton, 2013; Rossow, Sasaki, Digman, & Gratton, 2010
)
where the position of the observation volume is changed with respect to time. This
means that the correlation is dependent upon how fast the fluorescent molecules are
moving as well as how fast the observation volume is changing in a time-dependent
manner.
RICS can be used to detect and measure fluorescent dynamics in complex envi-
ronments such as the cellular cytoplasm and membrane organelles. While determi-
nation of protein oligomerization and protein complex stoichiometry is a challenging
task in live cells, it is an important one as many biological processes often depend
upon protein clustering (
Choi, Zareno, Digman, Gratton, & Horwitz, 2011; Digman,
Brown, Horwitz, Mantulin, & Gratton, 2008; Digman, Wiseman, Choi, Horwitz, &
Gratton, 2009
). Recently, determination of fluorescently labeled protein clustering
has become much more robust with number and brightness (N&B) analysis
(
Digman, Brown, et al., 2008, Digman, Dalal, Horwitz, & Gratton, 2008
). N&B
