UV-Vis spectroscopic measurements consist one of the most widely used methods for protein quantification. This is due to the fact that the tryptophan and tyrosine (and to a lesser extent the phenylalanine) residues of proteins strongly absorb at 280 nm. Hence, the corresponding absorbance is used for the calculation of protein concentration, based on specific quantification formulas. If the protein sample does not have tryptophan or tyrosine, the concentration can still be easily measured by the absorption at 205 nm in which the peptide bonds are analysed directly (Scopes method). Conformational studies can also be performed under special conditions.
The spectroscopic equipment of TPCI/NHRF accessible through the Instruct-EL hub includes a UV-Vis-NIR spectrophotometer (Lambda 19 by Perkin Elmer), which is a general-purpose instrument for absorption spectroscopy in the ultraviolet, visible, and near infrared spectral regions. It allows measurement of transmittance and reflectance of diffuse or scattering materials as a function of wavelength. Moreover, the optical scheme consists of a double beam double monochromator configuration, while the useable wavelength range is 250 – 2500 nm with temperature control. UV-Vis spectroscopic services are mainly focused towards protein quantification and concentration determination. Moreover, reflectance measurements of thin films and surfaces is also available.