ABSTRACT
We describe the photophysical properties of Seta-633, a commercially available near-infrared (NIR) dye, and its use as a fluorescent label to study the interaction between low-molecular-weight analytes and proteins using fluorescence lifetime as the readout parameter. In a model assay, we demonstrate that a biotinylated Seta-633 tracer binds to antibiotin with high specificity. Importantly, the lifetime of Seta-633-biotin increases about 1.8-fold upon binding to a specific antibody (antibiotin, MW = 160 kDa), while the titration with bovine serum albumin (BSA) or nonspecific antibody does not result in a noticeable change in lifetime. This behavior is contrary to that of fluorescent tracers like Cy5 or Alexa 647, which typically exhibit much smaller lifetime changes upon binding to antibodies.
Subject(s)
Fluorescent Dyes/chemistry , Proteins/chemistry , Antibodies/immunology , Biotin/immunology , Fluorescence , Half-Life , Infrared RaysABSTRACT
We describe the spectral properties of an amine-reactive, pH-sensitive, long-wavelength ratiometric fluorescent label having a pK(a) in the physiological pH range. The label exhibits its main absorption and emission in the near-infrared (NIR) region. On deprotonation, a blue shift of the excitation maximum is observed. Importantly, both the protonated and deprotonated forms of the label are fluorescent, with the deprotonated form having an extremely large Stokes shift of more than 100 nm. The spectral and photophysical properties of this pH label are compared with the properties of the protein-conjugated forms. Due to the observed pK(a) shift to the acidic pH range upon conjugation to proteins, such labels are ideal for studying phagocytic events and their regulation by drugs and/or environmental factors.
