Capillary electrophoretic protein separations in polyacrylamide-coated silica capillaries and buffers containing ionic surfactants

Strege, Mark A.; Lagu, Avinash L.

doi:10.1016/0021-9673(93)80470-s

Cited by 49 publications

(19 citation statements)

References 25 publications

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“…Buffers, therefore, must be carefully optimized in order to successfully separate proteins using MEKC. The selectivity of MEKC-based protein separations depends on the hydrophobic and electrostatic forces between the micelles and the surface of the protein [26,27]. These forces can be modified by varying the pH, ionic strength, and surfactant concentration to optimize the separation.…”

Section: Introductionmentioning

confidence: 99%

“…MEKC, introduced in 1984 by Terabe et al [24], has been used primarily for small-molecule analysis [25], although it has also been used for the analysis of macromolecules such as proteins [26][27][28][29][30][31][32]. Successful protein separations using MEKC, however, can be tricky as completely denatured proteins tend to adsorb a constant amount of SDS regardless of the acid-base chemistry or the hydrophobicity of the protein [33].…”

Section: Introductionmentioning

confidence: 99%

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Micellar electrokinetic chromatography of fluorescently labeled proteins on poly(dimethylsiloxane)‐based microchips

et al. 2006

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MEKC of standard proteins was investigated on PDMS microfluidic devices. Standard proteins were labeled with AlexaFluor(R) 488 carboxylic acid tetrafluorophenyl ester and filtered through a size-exclusion column to remove any small peptides and unreacted label. High-efficiency MEKC separations of these standard proteins were performed using a buffer consisting of 10 mM sodium tetraborate, 25 mM SDS, and 20% v/v ACN. A separation of BSA using this buffer in a 3.0 cm long channel generated a peak with a plate height of 0.38 microm in <20 s. Additional fast separations of myoglobin, alpha-lactalbumin, lysozyme, and cytochrome c also yielded peaks with plate heights ranging from 0.54 to 0.72 microm. All proteins migrated with respect to their individual pIs. To improve the separations, we used a PDMS serpentine chip with tapered turns and a separation distance of 25 cm. The number of plates generated increased linearly with increasing separation distance on the extended separation channel chips; however, the resolution reached an asymptotic value after about 7 cm. This limited the peak capacity of the separation technique to 10-12.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Micellar electrokinetic chromatography of fluorescently labeled proteins on poly(dimethylsiloxane)‐based microchips

et al. 2006

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“…Comparing protein peak efficiencies produced by the different capillary coatings investigated in this study, the highest peak efficiencies were obtained in the PHEAcoated capillary indicating minimal interactions between proteins and the polymer coating. The slower migration times and lower efficiencies obtained in LPA-coated capillaries are most likely due to weak interactions, primarily hydrogen bonding between proteins and LPA [42,43]. Reversible hydrogen bonding interactions between proteins and hydrophilic polymer coatings temporarily retain the proteins at the capillary surface, resulting in retardation of the analyte and in band broadening, similar to what happens in chromatographic separations.…”

Section: Protein-polymer Coating Interactionsmentioning

confidence: 90%

Poly‐N‐hydroxyethylacrylamide as a novel, adsorbed coating for protein separation by capillary electrophoresis

2003

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We present the polymer poly-N-hydroxyethylacrylamide (PHEA) (polyDuramide) as a novel, hydrophilic, adsorbed capillary coating for electrophoretic protein analysis. Preparation of the PHEA coating requires a simple and fast (30 min) protocol that can be easily automated in capillary electrophoresis instruments. Over the pH range of 3-8.4, the PHEA coating is shown to reduce electroosmotic flow (EOF) by about 2 orders of magnitude compared to the bare silica capillary. In a systematic comparative study, the adsorbed PHEA coating exhibited minimal interactions with both acidic and basic proteins, providing efficient protein separations with excellent reproducibility on par with a covalent polyacrylamide coating. Hydrophobic interactions between proteins and a relatively hydrophobic poly-N,N-dimethylacrylamide (PDMA) adsorbed coating, on the other hand, adversely affected separation reproducibility and efficiency. Under both acidic and basic buffer conditions, the adsorbed PHEA coating produced an EOF suppression performance comparable to that of covalent polyacrylamide coating and superior to that of adsorbed PDMA coating. The protein separation performance in PHEA-coated capillaries was retained for 275 consecutive protein separation runs at pH 8.4, and for more than 800 runs at pH 4.4. The unique and novel combination of hydrophilicity and adsorptive coating ability of PHEA makes it a suitable wall coating for automated microscale analysis of proteins by capillary array systems.

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“…To date only a few specific proteins or peptides were investigated [98][99][100][101][102]106], although this technique permits the analysis of several hundred polypeptides simultaneously in a short time, in a small volume, with high sensitivity [103]. Thus, CE-ESI-MS is a powerful alternative to common proteomic technologies.…”

Section: Ce-msmentioning

confidence: 99%

Differential polypeptide display: the search for the elusive target

Wittke

Kaiser

Mischak

2004

Journal of Chromatography B

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Capillary electrophoretic protein separations in polyacrylamide-coated silica capillaries and buffers containing ionic surfactants

Cited by 49 publications

References 25 publications

Micellar electrokinetic chromatography of fluorescently labeled proteins on poly(dimethylsiloxane)‐based microchips

Micellar electrokinetic chromatography of fluorescently labeled proteins on poly(dimethylsiloxane)‐based microchips

Poly‐N‐hydroxyethylacrylamide as a novel, adsorbed coating for protein separation by capillary electrophoresis

Differential polypeptide display: the search for the elusive target

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