Intrinsic Tryptophan Fluorescence (ITF)

Based on our brilliant technical scientists and a reliable and robust platform, Creative Biolabs can provide fluorescence assay relying on the intrinsic fluorescence of proteins, that arise from the excitation of aromatic amino acids, mainly tryptophan, to gain information about the structure and structural adjustments within proteins.

Intrinsic Tryptophan Fluorescence (ITF)

The intrinsic fluorescence constituents of proteins, predominantly derived from the aromatic amino acid tryptophan, which is present at concentrations of about 1 mol%. ITF can be emitted selectively excited at wavelengths between 295 and 305 nm. Consequently, most studies dealing with the stability of proteins predominantly refer to tryptophan emission when investigating intrinsic protein fluorescence to obtain fluorescence data that is measurable by fluorescence spectroscopy. Tryptophan dominates the emission of proteins and is the most sensitive to solvent polarity and the conformational changes in the local microenvironment of tryptophan, resulting in a fluorescence peak maximum shift and variation of the fluorescence intensity, fluorescence anisotropy, lifetime, and so on.

Fig.1 Absorption and emission spectra of tryptophan in water, pH 6.5, at T=25°C. (Garidel, 2008)

ITF based Biophysical Characterization Service

In principle, tryptophan fluorescence studies can utilize ITF of proteins to give a wealth of information regarding the location of the fluorophore within its macromolecular structure. The change of fluorescence intensity can strongly arise depending on the close surroundings of the change of intrinsic fluorescence of tryptophan residues, e.g., folding/unfolding, structural rearrangements, or binding of substances. Thus, Creative Biolabs can provide the genuinely label-free technique based on the ITF to monitor these fluorescence changes and fluorescence intensity or maximum emission wavelength (λmax) with high time-resolution for structural information of the macro-molecule and to investigate the proteins binding affinity and conformational changes in proteins.

Fig.2 Effect of pH (A), temperature (B), and ionic strength (C) on the emission spectra of tryptophan. (Garidel, 2008)

Typical Features of Our ITF Service

• Sensitive tryptophan fluorometric detection for protein analysis
• Coverage of an extensive protein concentration range from pM to mM
• Wide molecule size ranges from 1 kDa to 1 MDa
• Highly sensitive to conformational changes of a protein (favorable signal-to-noise ratios)
• Without modification of the protein and the need to spike an extrinsic fluorescence dye (label-free)
• Enjoy minimal protein sample consumption (in the range of 50 μg and below)
• Very short analysis time to enable high throughput
• Robust analysis of data quality and the simplicity of the procedure

Applications

• The information about the structure and function of proteins
• The conformational changes and the stability in protein structure
• The protein folding/unfolding, conformational rearrangements, and ligand binding
• In-depth feature analysis (influence of mutations, modifications, conjugations on protein stability and aggregation)
• The quick, material-saving, and reliable analytical means during early formulation development

With well-established biophysical characterization platform, the experienced scientists at Creative Biolabs are dedicated to using ITF with monitoring protein (un) folding, and investigating protein conformational changes, binding and interactions to promote drug discovery process. Our high-quality ITF biophysical characterization service will significantly contribute to the success of your projects. If you are interested in our service, please feel free to contact us for more details.

Reference

1. Garidel, P.; et al. A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy. Biotechnology Journal: Healthcare Nutrition Technology. 2008, 3: 1201-1211.

For Research Use Only.