Abstract
Fluorescence-based assays are dynamic, sensitive and robust quantitative methods for studying the conformational plasticity, enzyme kinetics, dynamics, and macromolecular interactions. However, the caveat for most of these experimental approaches is to label the target protein with one or more extrinsic fluorophores having required photophysical characteristics. Fluorescein isothiocyanate (FITC), due to its high quantum efficiency and conjugate stability, is most widely used fluorescence labelling reagent for such experimental approaches. However, the limitations in this method are requirement of high protein concentration, maintenance of protein stability during the entire labelling process as well as presence of significant background fluorescence due to ineffective removal of unreacted FITC. Therefore, to circumvent these setbacks, we have devised a novel strategy through introduction of tandem affinity purification tags at both the N- and C-termini of the protein of interest. Using this method, we have efficiently labelled target protein with significant decrease in precipitation, degradation and background fluorescence due to residual FITC. This facile and rapid technique may also be used as a basis for labelling procedures with other fluorophores and hence has a broad application in the field of biomedical sciences.
Figure 1