Determination of picomolar concentrations of proteins using novel amino reactive chameleon labels and capillary electrophoresis laser-induced fluorescence detection

✍ Scribed by Douglas B. Craig; Bianca K. Wetzl; Axel Duerkop; Otto S. Wolfbeis

• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 310 KB
• Volume: 26
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/elps.200410332

✦ Synopsis

Py-1 and Py-6 are novel amino-reactive fluorescent reagents. The names given to them reflect that they consist of a pyrylium group attached to small aromatic moieties. Upon reaction with a primary amine there is a large spectral shift in the reagent, rendering them effectively fluorogenic. In this study, these reagents were used to label a test protein, (human serum albumin), and the sample was analyzed by capillary electrophoresis and laser-induced fluorescence detection. Detection limits after a 60 min labeling reaction at 22 degrees C (Py-1) and 50 degrees C (Py-6) were 6.5 ng/mL (98 pM) for Py-1 and 1.2 ng/mL (18 pM) for Py-6. Separation of immunoglobulin G (IgG), human serum albumin, lipase, and myoglobin after labeling with Py-6 were performed. The method was further modified to make it amenable to automation. Unlike many other amino reactive reagents used to label protein amino groups, reaction with Py-1 and Py-6 do not alter the charge of the protein and the advantage of this with respect to electrophoretic separations is discussed.