Electrophoretic Separation of Proteins on a Microchip with Noncovalent, Postcolumn Labeling

Liu, Yingjie; Foote, Robert S.; Jacobson, Stephen C.; Ramsey, Roswitha S.; Ramsey, J. Michael

doi:10.1021/ac000625f

Cited by 136 publications

(127 citation statements)

References 33 publications

(60 reference statements)

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“…The sizing and quantitation data of these milk proteins are shown in Table 2. Except for casein aggregations, the sizing accuracy was within 3.5%, which was regarded as good in ME [4][5][6][7][8][9][10] and the quantitation results were also good compared to those of another report [19]. Consequently, these results suggested that our staining method was promising for analysis of real biological proteins.…”

Section: Application Of the Double-staining Methods To The Analysis Ofsupporting

confidence: 59%

“…To enhance the sensitivity, alternatives such as LIF detection instead of UV detection [7], on-line preconcentration [8], porous membrane [9] and a special detection method like refractive index detection [10] have been examined. However, they needed custom-made instruments and were labor intensive, and so have not been put to practical use.…”

Section: Introductionmentioning

confidence: 99%

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Highly sensitive double‐fluorescent dye staining on microchip electrophoresis for analysis of milk proteins

et al. 2008

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We demonstrated a highly sensitive double-fluorescent dye staining in microchip electrophoresis (ME) for analysis of milk proteins. The detection sensitivity of ME was very limited so far and needed improvement. Our staining method consisted of two steps. First, in sample preparation before electrophoresis, protein was covalently bound to an amine-reactive fluorescent dye, Cy5. Then, the Cy5-attached protein was denatured with SDS and was further stained, during electrophoresis, with Agilent fluorescent dye, which was noncovalently attached to hydrophobic regions of the SDS-protein complexes. This double-fluorescent staining enhanced fluorescent intensity and lowered the detection limit to 200 pg of protein. This provided higher sensitivity than silver-or SYPRO Ruby-staining methods, which have previously given the highest sensitivity in protein staining. In addition, we applied our staining method to analysis of milk proteins and achieved their successful detection, whereas it was difficult to analyze them by the unimproved method. Keywords:Double-fluorescent dye staining / Microchip electrophoresis / Potent milk allergen / Size-based protein separation

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Section: Application Of the Double-staining Methods To The Analysis Ofsupporting

confidence: 59%

Section: Introductionmentioning

confidence: 99%

Highly sensitive double‐fluorescent dye staining on microchip electrophoresis for analysis of milk proteins

et al. 2008

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“…Standard capillary separation techniques were transferred to the microchip format and LIF detection systems for the ultrasensitive detection of labeled proteins were developed. Using LIF detection in the visible (VIS) range, separation of covalently labeled proteins [5,6] and peptides [7,8] as well as postcolumn labeling for protein chip electrophoresis have been demonstrated [9,10]. Recently, a label-free interferometric backscatter method has been described by Wang et al [11], where proteins could be detected in the nM range.…”

Section: Introductionmentioning

confidence: 99%

Single cell manipulation, analytics, and label-free protein detection in microfluidic devices for systems nanobiology

et al. 2005

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Single cell analytics for proteomic analysis is considered a key method in the framework of systems nanobiology which allows a novel proteomics without being subjected to ensemble-averaging, cell-cycle, or cell-population effects. We are currently developing a single cell analytical method for protein fingerprinting combining a structured microfluidic device with latest optical laser technology for single cell manipulation (trapping and steering), free-solution electrophoretical protein separation, and (label-free) protein detection. In this paper we report on first results of this novel analytical device focusing on three main issues. First, single biological cells were trapped, injected, steered, and deposited by means of optical tweezers in a poly(dimethylsiloxane) microfluidic device and consecutively lysed with SDS at a predefined position. Second, separation and detection of fluorescent dyes, amino acids, and proteins were achieved with LIF detection in the visible (VIS) (488 nm) as well as in the deep UV (266 nm) spectral range for label-free, native protein detection. Minute concentrations of 100 fM injected fluorescein could be detected in the VIS and a first protein separation and label-free detection could be achieved in the UV spectral range. Third, first analytical experiments with single Sf9 insect cells (Spodoptera frugiperda) in a tailored microfluidic device exhibiting distinct electropherograms of a green fluorescent protein-construct proved the validity of the concept. Thus, the presented microfluidic concept allows novel and fascinating single cell experiments for systems nanobiology in the future.

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“…Glass microchips were further applied for the separation of fluorescently labeled proteins. Applications comprised the separation of labeled marker proteins with M concentrations and LIF detection (Liu et al, 2000a), an assay for theophylline in the therapeutically useful range (Chiem and Harrison, 1997), as well as a postcolumn labeling method with a mass detection limit of <0.5 pg for model proteins (Liu et al, 2000b).…”

Section: Introductionmentioning

confidence: 99%

Towards single molecule analysis in PDMS microdevices: from the detection of ultra low dye concentrations to single DNA molecule studies

Ros

Hellmich

Duong

et al. 2004

Journal of Biotechnology

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In this paper, we report on the performance of electrophoretical separation and laser-induced fluorescence (LIF) detection of dyes and fluorescently labeled biomolecules in poly(dimethylsiloxane) (PDMS) microdevices. The dyes fluorescein and fluorescein isothiocyanate (FITC) have been separated effectively in nM concentrations. Fluorescein injections gave linear concentration response in the range from 4 to 100 pM. As ultimate detection sensitivity, 100 fM injected fluorescein was obtained. Further, 100 fM injected fluorescein could be detected. This is to our knowledge the smallest electrokinetically injected dye concentration detected on a microchip. Injection studies of fluorescently labeled avidin revealed a theoretical detection limit of 25 nM for laser-induced fluorescence detection in good agreement with separations in glass chips. Furthermore, the injection of several and even one single DNA molecule using a PDMS cross injector has been demonstrated as well as free solution separation of -and T2-DNA (60 pM each) in periodically structured channels.

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Electrophoretic Separation of Proteins on a Microchip with Noncovalent, Postcolumn Labeling

Cited by 136 publications

References 33 publications

Highly sensitive double‐fluorescent dye staining on microchip electrophoresis for analysis of milk proteins

Highly sensitive double‐fluorescent dye staining on microchip electrophoresis for analysis of milk proteins

Single cell manipulation, analytics, and label-free protein detection in microfluidic devices for systems nanobiology

Towards single molecule analysis in PDMS microdevices: from the detection of ultra low dye concentrations to single DNA molecule studies

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