Capillary sodium dodecyl sulfate gel electrophoresis of proteins I. Reproducibility and stability

Shieh, Paul; Hoang, Dao; Guttman, András; Cooke, Nelson

doi:10.1016/0021-9673(94)80463-x

Cited by 43 publications

(18 citation statements)

References 14 publications

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“…Shieh et al identified a-lactalbumin and b-lactoglobulin by CSE/UV absorbance detection [22], while the molecular mass of casein was not shown. Chiu et al demonstrated LIF detection of proteins separated by CE using SYPRO red [23], where only bovine serum albumin and b-casein were detected.…”

Section: Application To Biological Samplesmentioning

confidence: 98%

Analysis of proteins in biological samples by capillary sieving electrophoresis with postcolumn derivatization/laser‐induced fluorescence detection

2011

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Previously, we have demonstrated postcolumn derivatization of proteins separated by capillary sieving electrophoresis (CSE), in which naphthalene-2,3-dicarbaldehyde was employed as a fluorogenic labeling reagent. Standard proteins separated by CSE were reacted with naphthalene-2,3-dicarbaldehyde in the presence of 2-mercaptoethanol (2-ME) which plays a role of a reducing agent in the derivatization reaction. To improve the sensitivity, we attempted the use of ethanethiol instead of 2-ME. Ethanethiol showed 1.4- to 4.5-fold lower limits of detection for proteins than 2-ME. Furthermore, we found that 8-aminopyrene-1,3,6-trisulfonate (APTS) is a good marker for relative electrophoretic mobilities of proteins in CSE. Since APTS is a fluorescent and trivalent anion, it generates strong fluorescence and migrates faster than any of the proteins. Therefore, we employed APTS as a marker to obtain the relative electrophoretic mobilities of proteins. The present method was applied to the analyses of proteins in biological samples. Human Ewing's family tumor cell line 'RDES' was used as a sample. The cultured cells were lysed with a buffer containing Tris-HCl, NaCl, sodium dodecyl sulfate, and 2-ME. After denaturation, the lysate was directly introduced into the capillary. Several peaks, which would correspond to proteins with molecular mass ranging from 10 to 93 kDa, were found in the cell lysate. In addition, we measured a milk sample by the CSE with postcolumn derivatization. The electropherogram showed five major peaks which corresponded to α-lactalbumin, β-lactoglobulin, κ-casein, bovine serum albumin, and mixture of α- and β-casein.

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Section: Application To Biological Samplesmentioning

confidence: 98%

Analysis of proteins in biological samples by capillary sieving electrophoresis with postcolumn derivatization/laser‐induced fluorescence detection

2011

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“…7 Consequently, PEO is a commercial sieving matrix for SDS CGE (e.g., Beckman SDS 14-200). 59,109 Unlike LPA, dextran, and other popular polymers, PEO is nonreactive with post-column uorogenic labelling reagents like NDA (naphthalene-2,3-dicarbaldehyde). 59 The separation power of 3% PEO SDS CGE has been reported as comparable with the performance of slab-gel SDS PAGE.…”

Section: Polyethylene Oxide (Peo)mentioning

confidence: 99%

Polymer sieving matrices in microanalytical electrophoresis

Chung

Kim

Herr

2014

Analyst

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Microfluidic design has advanced existing protein separation capabilities and supported novel assays. Key metrics for successful protein separations include fast, robust, and sensitive analysis of complex mixtures of bio-macromolecules. Attaining high separation resolution is a chief concern. Here we review recent advances in polymer-based electrophoresis sieving materials that are impacting microfluidic bioanalytical applications. Looking forward, we comment on unmet needs for advanced separation media in microto-nanoscale devices.

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“…and poly(vinyl) alcohol [25]. These authors have also used several techniques to determine molecular weights of proteins including plotting migration time against molecular weight standards [11- 30] and the use of Ferguson plots [12,16,21,22,24]. This paper will illustrate the development, optimization, and validation of a CGE method used to support commercial production of Synagis® (a monoclonal antibody to prevent Respiratory Syncytial Virus infection in high-risk infants).…”

Section: Introductionmentioning

confidence: 97%

Optimization, Validation, and Use of Capillary Gel Electrophoresis for Quality Control Testing of Synagis®, a Monoclonal Antibody

Schenerman¹,

Bowen²

2001

CE in Biotechnology: Practical Applications for Protein and Peptide Analyses

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Capillary electrophoresis methods offer many advantages over their conventional electrophoresis counterparts including: greater precision, higher throughput, on-line detection and less waste. Capillary gel electrophoresis (CGE), using replaceable sieving polymers, is Widely used as an alternative to SDS-PAGE and densitometry CGE offers greater automation capabilities than SDS-PAGE and can be operated using conventional chromatography system software. This software for handling and analyzing data makes the validation of CGE methods look quite similar to the validation of an HPLC method. The same parameters can be examined, including: precision, accuracy, linearity, selectivity, range, robustness, and system suitability. There are, however, certain key elements that are critical to a successful CGE method validation. The CGE quantification method must be shown to be equivalent to SDS-PAGE with densitometry and should be presented such that the densitometry plots look like the electropherograms. In addition, the sensitivity of the methods must be compared and the CGE method must have comparable or better sensitivity to SDS-PAGE with Coomassie staining. The validation must also be performed using materials that might be tested during the course of a typical process (in-process samples) in their particular matrices. The other vital element is assuring that acceptance criteria for the validation have been set to reasonable ranges. These ranges must be determined in pilot studies that are executed prior to the validation studies. Under most CGE conditions, the acceptance criteria will be quite similar to those seen in HPLC validations. Overall, CGE methods can be validated to the same level of assurance as HPLC methods. The CGE method described here has been approved by the FDA and is used routinely for testing of Synagis®.

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Capillary sodium dodecyl sulfate gel electrophoresis of proteins I. Reproducibility and stability

Cited by 43 publications

References 14 publications

Analysis of proteins in biological samples by capillary sieving electrophoresis with postcolumn derivatization/laser‐induced fluorescence detection

Analysis of proteins in biological samples by capillary sieving electrophoresis with postcolumn derivatization/laser‐induced fluorescence detection

Polymer sieving matrices in microanalytical electrophoresis

Optimization, Validation, and Use of Capillary Gel Electrophoresis for Quality Control Testing of Synagis®, a Monoclonal Antibody

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