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A new weakly basic amino‐reactive fluorescent label for use in isoelectric focusing and chip electrophoresis
Author(s) -
Schulze Philipp,
Link Martin,
Schulze Marcel,
Thuermann Sebastian,
Wolfbeis Otto S.,
Belder Detlev
Publication year - 2010
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201000007
Subject(s) - fluorescence , chemistry , electrophoresis , analyte , conjugated system , chromophore , isoelectric point , isoelectric focusing , chromatography , photochemistry , biochemistry , organic chemistry , physics , quantum mechanics , enzyme , polymer
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≈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 p I of proteins compared with common anionic labels, e.g. FITC. p I 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.