In situ determination of secretory kinase Fam20C from living cells using fluorescence correlation spectroscopy

Secretory proteins constitute a biologically crucial subset of proteins for regulation of some pathological and physiological processes, and they have become very important biomarkers in clinical diagnosis and therapeutic targets. So far, secretory protein functions and mechanisms have not been fully understood due to methodological limitations in detection of low-abundance proteins against medium background. Here, we propose a strategy to determine secretory protein from living cells in situ using fluorescence correlation spectroscopy (FCS). In this study, the recombinant protein Fam20C with SNAP-tag was used as a model protein, and O-6-benzylguanine (BG) derivatives bearing fluorescent dye as probes. We synthesized three fluorescent probes and investigated their fluorescent properties and diffusion behaviors in solution, and found the probe BG-Bodipy-561 more suitable for in situ labeling of Fam20C. We confirmed the specific binding of the probe to the target protein by combining FCS and in-gel fluorescence scanning methods. We studied the effects of some factors of the secretory Fam20C, and found that RNA interference significantly inhibited the synthesis of secretory fused Fam20C, and myriocin had no significant effect on the expression of secretory Fam20C, which indirectly illustrated that sphingolipid signaling can regulate the Fam20C activity. We believe that FCS is a very promising method to analyze secretory proteins from living cells in situ.