A new weakly basic amino-reactive fluorescent label for use in isoelectric focusing and chip electrophoresis

Abstract

In this study, we present a novel amino-reactive fluorescence marker (referred to as UR-431), which is well suited for electrophoretic techniques. A main feature of this marker is its weakly basic behavior when conjugated to analytes. Labeled primary amines exhibit a positive net charge and accordingly a cathodic mobility below a pH of 2.4. The label features a pH-independent fluorescence emission and is thus very interesting for electrophoretic applications such as IEF. The absorption maximum of this yellow daylight chromophore is at 431 nm, whereas fluorescence emission peaks at 537 nm (quantum yield approximately 0.1). The label was successfully conjugated to amines, peptides and proteins and separated via CE and MCE. The on-chip detection limit of labeled lysine using a mercury-lamp-based fluorescence microscope was determined as 12 nM. An important feature of the new label is that it effects only a subtle change of the pI of proteins compared with common anionic labels, e.g. FITC. pI values of proteins were investigated by comparing native proteins and labeled proteins in CIEF. UR-431 was also applied to sensitive detection of amines and peptides in MCE.