Protocol abstract
Nature Protocols 2, - 2842 - 2856 (2007)
Published online: 1 November 2007 | doi:10.1038/nprot.2007.410
Subject Categories: Biochemistry and protein analysis | Genomics and proteomics | Spectroscopy and structural analysis
Practical guidelines for dual-color fluorescence cross-correlation spectroscopy
Kirsten Bacia1,2 & Petra Schwille1
Abstract
Dual-color fluorescence cross-correlation spectroscopy (FCCS) allows for the determination of molecular mobility and concentrations and for the quantitative analysis of molecular interactions such as binding or cleavage at very low concentrations. This protocol discusses considerations for preparing a biological system for FCCS experiments and offers practical advice for performing FCCS on a commercially available setup. Although FCCS is closely related to two-color confocal microscopy, critical adjustments and test measurements are necessary to establish successful FCCS measurements, which are described in a step-by-step manner. Moreover, we discuss control experiments for a negative cross-correlation artifact, arising from a lack of detection volume overlap, and a positive artifact, arising from cross-talk. FCCS has been applied to follow molecular interactions in solutions, on membranes and in cells and to analyze dynamic colocalization during intracellular transport. It is a technique that is expected to see new applications in various fields of biochemical and cell biological research.
- BIOTEC, Department of Biophysics, Dresden University of Technology, Tatzberg 47-51, D-01307 Dresden, Germany.
- Present address: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720-3202, USA.
Correspondence to: Petra Schwille1 e-mail: petra.schwille@biotec.tu-dresden.de
nature-products
A-Z product listing
- Axiovert 100 microscope stand(Zeiss)
- inverted Axiovert 200(Zeiss)
- ITW Texwipe Clean Tips(Carl Zeiss)
- Power meter(Newport)
- spectroscopy grade isopropanol(Carl Zeiss)
- Whatman Lens Cleaning Tissue 105(Carl Zeiss)
