Quality Control Of FITC-labeled Proteins for Interactomics Investigations Using SDS Capillary Gel Electrophoresis and SPR Biosensor

Abstract

The technology of dye-labeled proteins has many fields of application, especially in interactomics. The aim of this work was to adapt protocol of conjugation of low molecular weight (12 – 15 kDа) heme-containing proteins with fluorescein isothiocyanate, isomer I, (FITC) for subsequent protein-protein interaction studies. We have monitored the quality of FITC-labeling of the target protein and comparative assessment of its binding capacity. Using the cytochrome C (Mw 12 kDа) as an example, it has been shown that using the three step method approach including conventional spectrophotometry, capillary gel electrophoresis and SPR analysis it is possible to assess: (i) the capability of the FITC-labeled target protein to interact with its protein partner and protein material from tissue lysates, (ii) the fact of dye conjugation with the protein, and (iii) the quality of purification for final protein preparation from unreacted free dye molecules