Practical guidelines for dual-color fluorescence cross-correlation spectroscopy
- 1 November 2007
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature Protocols
- Vol. 2 (11), 2842-2856
- https://doi.org/10.1038/nprot.2007.410
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.Keywords
This publication has 53 references indexed in Scilit:
- Oligomerization of the EGF Receptor Investigated by Live Cell Fluorescence Intensity Distribution AnalysisBiophysical Journal, 2007
- Investigation of the Dimerization of Proteins from the Epidermal Growth Factor Receptor Family by Single Wavelength Fluorescence Cross-Correlation SpectroscopyBiophysical Journal, 2007
- Diffusion and Segmental Dynamics of Double-Stranded DNAPhysical Review Letters, 2006
- Dual-Color Photon Counting Histogram Analysis of mRFP1 and EGFP in Living CellsBiophysical Journal, 2006
- One-step analysis of protein complexes in microliters of cell lysate using indirect immunolabeling & fluorescence cross-correlation spectroscopyNature Protocols, 2006
- A guide to choosing fluorescent proteinsNature Methods, 2005
- Two‐Hybrid Fluorescence Cross‐Correlation Spectroscopy Detects Protein–Protein Interactions In VivoChemphyschem, 2005
- Characterizing observation volumes and the role of excitation saturation in one-photon fluorescence fluctuation spectroscopyJournal of Biomedical Optics, 2005
- Dual‐Color Fluorescence Cross‐Correlation Spectroscopy Using Single Laser Wavelength ExcitationChemphyschem, 2004
- Art and Artefacts of Fluorescence Correlation SpectroscopyCurrent Pharmaceutical Biotechnology, 2004