Publication details
Low-molecular-mass fluorescent isoelectric focusing markers suitable for the effective tracking of pH gradient
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| Year of publication | 2023 |
| Type | Conference abstract |
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| Description | Isoelectric point (pI) markers are crucial internal standards used in isoelectric focusing (IEF) methods. Our laboratory has already introduced colored (UV and visible region absorbing) low-molecular-mass (LMM) pI markers for capillary isoelectric focusing (cIEF) as an alternative to the commonly used peptide and protein pI markers. The cIEF instrumentation can be coupled with on-line laser induced fluorescence (LIF) detection, which significantly increases detection sensitivity. However, there was considerable lack of suitable pI markers for this type of application. Consequently, our laboratory devised LMM fluorescein-based pI markers, initially covering narrow pH range (from 5.4 to 6.6). By adding various functional groups, more than 60 further candidate ampholytes covering broad range of pH have been synthetized since the original publication. The pI values of the candidate structures were then analyzed by conductometric titration or slab gel IEF, followed by cIEF-LIF analysis. Using the cIEF-LIF method enabled very precise pI calibration with the standard deviation reaching a value of a few hundredths of a pH unit (n=3). Several potential markers that contained impurities originating from the synthesis as well as some of the compounds that exhibited low fluorescence intensity were excluded from the set. Additionally, some of these have been purified by a newly developed cIEF-fractionation method. Total of 25 selected markers were calibrated using our previously published nitrophenol-based LMM pI markers. Four calibrated fluorescing pI markers evenly covering pH region of 8.8 to 4.0 were then applied for cIEF-LIF analysis and pI determination of referent fluorescently labeled human monoclonal IgG. |