Capillary sodium dodecyl sulfate gel electrophoresis of proteins II. On the Ferguson method in polyethylene oxide gels

Guttman, András; Shieh, Paul; Lindahl, J. L.; Cooke, Nelson

doi:10.1016/0021-9673(94)80464-8

Cited by 38 publications

(25 citation statements)

References 15 publications

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“…Figure 3A shows that the separation of eight proteins by SDS-CGE at 233 V/cm was accomplished using 3.5% PVA solution. This result is comparable to that employed for the separation of SDS-protein complexes using a coated capillary (in the absence of EOF) [36,37]. Without the regeneration of high bulk EOF, the RSD of their migration times was less than 0.7% (n = 10).…”

Section: Size Separation Of Proteins In the Presence Of Eofsupporting

confidence: 84%

“…In SDS-CGE analysis, capillaries are often coated to suppress EOF and minimize protein adsorption on the inner surface of the capillary [36,37]. However, in the case of multiplexed CE, the use of coated capillaries is not practical due to high costs and reduced ruggedness as compared to uncoated capillaries.…”

Section: Size Separation Of Proteins In the Presence Of Eofmentioning

confidence: 99%

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On‐line concentration of proteins by SDS‐CGE with LIF detection

Chang

Tseng

2008

Electrophoresis

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We present a simple approach for on-line concentration of SDS-protein complexes by using poly(vinyl alcohol) (PVA) solution in CGE. In comparison to the coated capillary, the presence of EOF in CGE omitted the need to fill the capillaries with polymer solutions prior to the analysis. More importantly, we found that highly reproducible separation of eight proteins by 3.5% PVA was achieved between runs and without the regeneration of high bulk EOF; the RSD of migration times was less than 0.7%. To further improve the concentration sensitivity, neutral PVA was introduced into the capillary with the help of EOF to act as sieving matrix. The occurrence of stacking at the boundary between the PVA and the sample zone is mainly due to the retardation of proteins by PVA. As a result, the LODs at an S/N of 3 for SDS-protein complexes are of the order of sub-nM to several nM. For example, the LOD for BSA is 0.78 nM, which is a 91-fold sensitivity enhancement over the normal injection. In addition, our stacking method has been applied to the analyses of proteins in Escherichia coli cells. The peak for b-galactosidase (E. coli) was observed after 0.1 mM b-galactosidase was spiked into the E. coli samples.

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Section: Size Separation Of Proteins In the Presence Of Eofsupporting

confidence: 84%

Section: Size Separation Of Proteins In the Presence Of Eofmentioning

confidence: 99%

On‐line concentration of proteins by SDS‐CGE with LIF detection

Chang

Tseng

2008

Electrophoresis

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“…The Ferguson plot of protein mobilities was frequently used in PAGE in absence of ionic surfactants [56], nevertheless its use was proposed also for SDS CSE [33,34]. It shows a relationship between the protein mobility and the concentration of a sieving medium.…”

Section: Resultsmentioning

confidence: 99%

“…Significant deviations from the literature data were observed for a number of proteins [46]. To improve the accuracy of the M r determination, it was suggested to use the Ferguson method [33,34]. It improved the accuracy of M r for some proteins but, e.g., for IgG L-chain, where SDS CSE originally showed 87% error, the Ferguson method did not get below 17% error [34].…”

Section: Introductionmentioning

confidence: 99%

Size separation of proteins by capillary zone electrophoresis with cationic hitchhiking

Dolnı́k¹,

Gurske²

2011

Electrophoresis

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The paper describes a method of size separation of proteins by capillary sieving electrophoresis with cationic surfactant. Proteins are separated within 12 minutes with repeatability of migration times better than 0.2%. Some proteins achieve the separation efficiency of 200,000 theoretical plates. The method can be used for determination of protein relative molecular masses. The accuracy of the determined relative molecular masses and the limitation of the method were investigated by the analysis of more than 60 proteins. The method also allows separation of protein oligomers. Proteins can be quantitated after the electrokinetic injection in the concentration range 0.07–0.43 g/L. The average detection limit is about 2 mg/L.

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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 II. On the Ferguson method in polyethylene oxide gels

Cited by 38 publications

References 15 publications

On‐line concentration of proteins by SDS‐CGE with LIF detection

On‐line concentration of proteins by SDS‐CGE with LIF detection

Size separation of proteins by capillary zone electrophoresis with cationic hitchhiking

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

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