Protein Sizing on a Microchip

Bousse, Luc; Mouradian, Stéphane; Minalla, Abdel; Yee, Herman; Williams, Kathi; Dubrow, Robert

doi:10.1021/ac0012492

Cited by 277 publications

(266 citation statements)

References 44 publications

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“…Several applications of the bioanalyzer system have been published [9][10][11][12]. Smith et al [9] used the Agilent 2100 bioanalyzer to detect telomerase expressed in the prostatic fluid of patients being evaluated for prostate cancer.…”

Section: Introductionmentioning

confidence: 99%

Detection of DNA mutations associated with mitochondrial diseases by Agilent 2100 bioanalyzer

Lu¹,

Tso²,

Yang³

et al. 2002

Clinica Chimica Acta

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Background: Molecular analysis of mitochondrial DNA (mtDNA) has provided a final diagnosis for many of the mitochondrial diseases. We evaluated the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA) to determine whether the system could replace the conventional restriction fragment length polymorphism (RFLP) analysis by the agarose gel electrophoresis for the detection of the mtDNA mutation. Methods: Three members of a family with MELAS syndrome and four members of a family with MERRF syndrome were recruited for this study. After PCR and restriction enzyme digestion, DNA fragments were separated on the Agilent 2100 bioanalyzer in conjunction with the DNA 500 and DNA 1000 Labchip kits and by electrophoresis on precast 3% agarose gels. Results: The data generated by the DNA 500 and DNA 1000 assays using the Agilent 2100 bioanalyzer showed a lower percentage error and a better reproducibility as compared to those obtained by the conventional method. Conclusion: Based on the performance of the bioanalyzer, we suggest that this novel Labchip is adequate to replace the current RFLP analysis by the agarose gel electrophoresis for mtDNA mutation detection. D

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Section: Introductionmentioning

confidence: 99%

Detection of DNA mutations associated with mitochondrial diseases by Agilent 2100 bioanalyzer

Lu¹,

Tso²,

Yang³

et al. 2002

Clinica Chimica Acta

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“…The AFD attached to the micelles emits highly fluorescent background interfering with the detection of SDS-protein complexes. To overcome the problem of the background, dilution channels were added just before the detection point of the Bioanalyzer to decrease the concentration of the micelles below their critical concentration and then a dilution step suppressed the background [5]. In this dilution step, the fluorescent intensity of SDS-protein complexes as well as that of micelles was simultaneously reduced due to a) The data for LOD were averaged from ten different measurements using ten different chips.…”

Section: Sensitivity Of Double-staining Methodsmentioning

confidence: 99%

“…Therefore, in the Bioanalyzer, conventionally, non-Cy5-labeled protein is just stained with AFD, which emits fluorescence after binding to the hydrophobic regions (long alkyl chains of dodecyl sulfate) of SDS-protein complexes [5]. Therefore, the single staining was performed.…”

Section: On-separation Fluorescent Dye Stainingmentioning

confidence: 99%

“…Successful highperformance electrophoretic separations have been done on microchips. For example, Yao et al [4] compared the performance of CE-dependent SBPS to that of glass-based ME and Bousse et al [5] succeeded in separating 11 proteins from 6 to 200 kDa on a soda-lime glass microchip having channels filled with a low-viscosity separation medium, poly(dimethyl acrylamide), as a substitute for poly(-acrylamide) gel.…”

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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“…8,[18][19][20] Presumably due to the low binding efficiency and high background noise, only moderate low LODs (30−500 ng/mL) were achieved. 8,20 In this paper, we introduce a novel strategy to stain proteins for capillary SDS-PAGE. Figure 1 presents a schematic presentation of this novel approach.…”

mentioning

confidence: 99%

Staining Method for Protein Analysis by Capillary Gel Electrophoresis

Lü

Wang

et al. 2007

Anal. Chem.

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A novel staining method and the associated fluorescent dye were developed for protein analysis by capillary SDS-PAGE. The method strategy is to synthesize a pseudo-SDS dye and use it to replace some of the SDS in SDS-protein complexes so that the protein can be fluorescently detected. The pseudo-SDS dye consists of a long, straight alkyl chain connected to a negative charged fluorescent head and binds to proteins just as SDS. The number of dye molecules incorporated with a protein depends on the dye concentration relative to SDS in the sample solution, since SDS and dye bind to proteins competitively. In this work, we synthesized a series of pseudo-SDS dyes, and tested their performances for capillary SDS-PAGE. FT-16 (a fluorescein molecule linked with a hexadodecyl group) seemed to be the best among all the dyes tested. Although the numbers of dye molecules bound to proteins (and the fluorescence signals from these protein complexes) were maximized in the absence of SDS, high-quality separations were obtained when co-complexes of SDS-proteindye were formed. The migration time correlates well with protein size even after some of the SDS in the SDS-protein complexes was replaced by the pseudo-SDS dye. Under optimized experimental conditions and using a laser-induced fluorescence detector, limits of detection of as low as 0.13 ng/ mL (bovine serum albumin) and dynamic ranges over 5 orders of magnitude in which fluorescence response is proportional to the square root of analyte concentration were obtained. The method and dye were also tested for separations of real-world samples from E. coli.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is one of the most commonly used methods for protein analysis. Usually, a staining and destaining process is involved in order to properly detect the separated protein bands. Capillary electrophoresis (CE), and more recently microchip-based electrophoresis, is a miniaturized electrophoresis technique that has many advantages (e.g., high separation efficiency, short separation time, low mass detection limit, on-line detection, and automated operation) over traditional slab gel techniques. SDS-PAGE has been performed in capillaries 1-5 and on microchips. 6-9 Unfortunately, the staining and destaining process used in conventional slab gels cannot be simply adopted in CE. Typically, the separated proteins are detected in-column by either an ultraviolet (UV) absorbance or a laser-induced fluorescence (LIF) detection system. UV absorption is arguably the most frequently used detection mode in CE. It is also commonly employed in capillary SDS-PAGE, 1,2 since protein-SDS complexes absorbs light around 280 nm due to the aromatic side groups of amino acids and around 200−220 nm due to the peptide bonds between amino acids. LIF detection is preferred when low limit of detection (LOD) and wide linear dynamic range are desired. Native fluorescence of proteins has been explored for direct protein detection, 10 but it is not widely accepted because of the use of expe...

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Protein Sizing on a Microchip

Cited by 277 publications

References 44 publications

Detection of DNA mutations associated with mitochondrial diseases by Agilent 2100 bioanalyzer

Detection of DNA mutations associated with mitochondrial diseases by Agilent 2100 bioanalyzer

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

Staining Method for Protein Analysis by Capillary Gel Electrophoresis

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