Fluorescein-based isoelectric point markers as a tool for tracking of pH gradient in highly sensitive isoelectric focusing analysis with laser induced fluorescence detection

0584029 - ÚIACH 2025 DE eng A - Abstract
Dadajová, Pavlína - Duša, Filip - Čmelík, Richard - Šlais, Karel
Fluorescein-based isoelectric point markers as a tool for tracking of pH gradient in highly sensitive isoelectric focusing analysis with laser induced fluorescence detection.
CE Forum 2024. Aalen: Aalen University, 2024. s. 22-22.
[CE Forum 2024. 28.02.2024-29.02.2024, Aalen]
R&D Projects: GA ČR(CZ) GA23-04703S
Institutional support: RVO:68081715
Keywords : capillary isoelectric focusing * laser induced fluorescence (LIF) detection * fluorescein-based pI markers
OECD category: Analytical chemistry

The capillary isoelectric focusing (cIEF) instrumentation can be coupled with on-line laser induced fluorescence (LIF) detection, which significantly increases detection sensitivity. However, there was considerable lack of any suitable markers of isoelectric point (pI) for this type of application. In 2002 our laboratory presented four fluorescein-based pI markers initially covering narrow pH range (from 5.4 to 6.6). Since then, by adding various functional groups, more than 60 further candidate ampholytes covering broad range of pH have been synthetized and tested. However, some of the nonconforming compounds exhibiting low fluorescence intensity or containing high portion of synthesis by-products, were excluded from the set. 22 markers were identified as suitable for the cIEF-LIF method. Finally, four calibrated pI markers, evenly covering the whole length of the pH gradient from 4.05 to 8.73, were selected and further applied for cIEF-LIF analysis and pI determination of fluorescently labeled antibody (IgG-FITC) and cell lysate from Spirulina sp., containing phycobiliproteins with naturally occurring fluorescence.
Permanent Link: https://hdl.handle.net/11104/0352030