Seeing the Wood through the Trees: A Review of Techniques for Distinguishing Green Fluorescent Protein from Endogenous Autofluorescence
✍ Scribed by Nicholas Billinton; Andrew W. Knight
- Book ID
- 102566632
- Publisher
- Elsevier Science
- Year
- 2001
- Tongue
- English
- Weight
- 450 KB
- Volume
- 291
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
The green fluorescent protein (GFP) 2 from the jellyfish Aequorea victoria has recently leapt to prominence within numerous biological fields. Interest in the use of GFP has grown enormously over the past 5 years. The number of papers concerning GFP held in the Web of Science database, which abstracts the scientific literature (http://wos.mimas.ac.uk), rose from 13 in the period 1981-1994, to over 3400 in the period 1995 to October 2000. This is due to the ability to clone and heterologously express GFP genes in a diverse range of cells and organisms, from bacteria and yeast, to plants and mammals, coupled with favorable properties such as high stability, minimal toxicity, noninvasive detection, and the ability to generate the highly visible fluorophore in vivo in the absence of external cofactors. Thus, GFP has become a truly versatile marker for visualizing physiological processes, monitoring subcel-lular protein localization, distinguishing successful transfection, or reporting on gene expression. As such a powerful tool, GFP now impinges on almost every area of biological research.
However, unless GFP is very highly expressed or densely localized, the fluorescence signal from GFP will invariably be contaminated with endogenous cellular or media fluorescence. Researchers often think they have failed in effectively using GFP as a marker, when actually they have succeeded. Their real problem was simply the detection of GFP in the sea of other fluorescent species within biological material, which fluoresce at a similar wavelength. Cells in most organisms exhibit a natural fluorescence, commonly called "autofluorescence," from a range of species including metabolites and structural components. This has been characterized in such diverse examples as human epidermal cells (1), rat hepatocytes (2), Drosophila melanogaster (3), numerous fungi (4 -6), and plants such as maize (7) and alfalfa (8). Many cellular metabolites exhibit autofluorescence, and since cellular extracts are often crucial components of culture media, such media can also be intensely autofluorescent, compounding the problem.
Autofluorescence spectra are generally broad and encompass most of the visible spectral range, overlapping the emission wavelengths of green fluorescent protein and its many derivatives. When measuring GFP, the presence of autofluorescence often leads to