Polymer sieving matrices in microanalytical electrophoresis

Chung, Minsub; Kim, Dohyun; Herr, Amy E.

doi:10.1039/c4an01179a

Cited by 37 publications

(36 citation statements)

References 174 publications

(376 reference statements)

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“…Nevertheless, for over 4 decades, slab polyacrylamide gel electrophoresis (PAGE) has remained ubiquitous for protein analysis. 8 The polyacrylamide (PA) gel is a sieve that retards protein electromigration on the basis of size and structure, yielding protein separations that address questions in research and clinical laboratories. 9,10 …”

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confidence: 99%

Fabrication of an Open Microfluidic Device for Immunoblotting

et al. 2017

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Given the wide adoption of polydimethylsiloxane (PDMS) for the rapid fabrication of microfluidic networks and the utility of polyacrylamide gel electrophoresis (PAGE), we develop a technique for fabrication of PAGE molecular sieving gels in PDMS microchannel networks. In developing the fabrication protocol, we trade-off constraints on materials properties of these two polymer materials: PDMS is permeable to O2 and the presence of O2 inhibits the polymerization of polyacrylamide. We present a fabrication method compatible with performing PAGE protein separations in a composite PDMS-glass microdevice, that toggles from an “enclosed” microchannel for PAGE and blotting to an “open” PA gel lane for immunoprobing and readout. To overcome the inhibitory effects of O2, we coat the PDMS channel with a 10% benzophenone solution, which quenches the inhibiting effect of O2 when exposed to UV, resulting in a PAGE-in-PDMS device. We then characterize the PAGE separation performance. Using a ladder of small-to-mid mass proteins (Trypsin Inhibitor (TI); Ovalbumin (OVA); Bovine Serum Albumin (BSA)), we observe resolution of the markers in <60 s, with separation resolution exceeding 1.0 and CVs of 8.4% for BSA-OVA and 2.4% for OVA-TI, with comparable reproducibility to glass microdevice PAGE. We show that benzophenone groups incorporated into the gel through methacrylamide can be UV-activated multiple times to photocapture protein. PDMS microchannel network is reversibly bonded to a glass slide allowing direct access to separated proteins and subsequent in situ diffusion-driven immunoprobing and total protein Sypro red staining. We see this PAGE-in-PDMS fabrication technique as expanding the application and use of microfluidic PAGE without the need for a glass microfabrication infrastructure.

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Fabrication of an Open Microfluidic Device for Immunoblotting

et al. 2017

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“…Given the aforementioned drawbacks of crosslinked hydrogels, such as bubble formation, various polymer solutions have been employed as sieving networks. 31 While the replaceable polymer solutions guarantee the reuse of the capillary column with high run-to-run precision, the separation resolution is compromised because the weak intermolecular interactions among polymers render less rigid networks. In addition, highly viscous polymer solutions, such as linear polyacrylamide, require high pressure (1000 psi) to fill the column.…”

Section: •2 Molecular Sievingmentioning

confidence: 99%

“…This is impossible for some micro fluidic devices, which can only stand moderate pressures (<200 psi). 31 To this extent, compared with SGE, the CGE and MGE are more likely to require a facilitation of operation from sieving matrixes. 20 The electric current, as well as the electric field strength, can be controlled by the crosssection area of the buffer channel.…”

Section: •2 Molecular Sievingmentioning

confidence: 99%

Hydrogels in Electrophoresis: Applications and Advances

2020

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“…An established approach of CE is CE-SDS, which can be described as the translation of SDS-PAGE into the capillary format [4]. In contrast to typical polyacrylamide gels used for SDS-PAGE, in CE-SDS non-cross-linked entangled polymer networks are used as separation matrices, e.g., dextran, polyvinyl alcohol, polyethylene oxide, and polyethylene glycol [5][6][7][8].…”

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confidence: 99%

A comparative study of CE‐SDS, SDS‐PAGE, and Simple Western: Influences of sample preparation on molecular weight determination of proteins

et al. 2020

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The development of capillary electrophoresis, especially CE‐SDS devices, has led CE‐SDS to become an established tool in a wide range of applications in the analysis of biopharmaceuticals and is increasingly replacing its method of origin, SDS‐PAGE. The goal of this study was to evaluate the comparability of molecular weight (MW) determination especially by CE‐SDS and SDS‐PAGE. For ensuring comparability, model proteins that have little or no posttranslational modifications and an IgG antibody were used. Only a minor influence of sample preparation conditions, including sample buffer, temperature conditions, and different reducing agents on the MW determination were found. In contrast, the selection of the MW marker plays a decisive role in determining the accurate apparent MW of a protein. When using different MW markers, the deviation in MW determination can exceed 10%. Interestingly, CE‐SDS and 10% SDS‐PAGE hardly differ in their trueness of MW determination. The trueness in relation to the reference MW for each protein was calculated. Although the trueness values for the model proteins considered range between 1.00 and 1.11 using CE‐SDS, they range between 0.93 and 1.03 on SDS‐PAGE, depending on the experimental conditions chosen.

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Polymer sieving matrices in microanalytical electrophoresis

Cited by 37 publications

References 174 publications

Fabrication of an Open Microfluidic Device for Immunoblotting

Fabrication of an Open Microfluidic Device for Immunoblotting

Hydrogels in Electrophoresis: Applications and Advances

A comparative study of CE‐SDS, SDS‐PAGE, and Simple Western: Influences of sample preparation on molecular weight determination of proteins

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