|Figure 1. Green fluorescence in NIH3T3 cells expressing GFP.|
Characteristics of GFP such as the fluorescence color and intensity can be altered by changing the amino acid residues around the chromophore . These mutations of GFP provide a number of color variants such as blue and yellow. These variations of GFP can be used to construct fluorescent chimeric proteins to be expressed in living cells, tissues, and entire organisms, after transfection with engineered vectors. Other colors such as red fluorescent proteins have been isolated from other species, including coral reef organisms, and are used in assays requiring a fluorescent protein with different characteristics than GFP. Other alterations have been made to improve protein folding and chromophore formation. Besides amino acid substitutions, many other silent mutations have been made to the wild type sequence. Codon changes in the DNA sequence for example, can reflect the intended host’s codon bias.
The small size of fluorescent proteins, along with their multitude of colors and wavelengths, have made this tool ideal for BOTH imaging and non-imaging uses. Chimeric proteins can be tracked using imaging to determine cellular protein tracking and localization. Transfection efficiency can be ascertained using a GFP tracer (see Figure 2). With increasing efficiency cultures normalized for cell number will exhibit increased fluorescence. Fluorescent protein tracers used with induced pluripotent stem cells (iPSCs) can be used to ascertain differentiation status. Non-imaging proximity assays such as LanthaScreen® or BRET use green fluorescent protein as an energy transfer acceptor molecule in numerous quantitative cellular assays.
Figure 2. Fluorescent signal as a function of GFP transfection efficiency.
Green fluorescent proteins and their multiple variations are extremely valuable tools in cellular assays and molecular imaging. They have helped to further our knowledge in many fields including signaling applications, proton transfer between proteins, in FRET/FLIM microscopy and in many other areas helping to understand cellular and protein structure and function. With the advent of new mutants and the discovery of new fluorescent proteins there seems to be no limit to the utility of these small proteins.
Do you use fluorescent proteins in your research? If so what protein(s) do you use?
By: BioTek Instruments, Paul Held PhD, Laboratory Manager