Polyethylene glycol significantly enhances the transfer of membrane immunoblotting

Zeng, Chi; Suzuki, Yuji; Alpert, Elliot

doi:10.1016/0003-2697(90)90107-k

Cited by 12 publications

(2 citation statements)

References 33 publications

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“…Therefore, preventing this adsorption is quite challenging but crucial for developing flow assays with enhanced sensitivity, multiplexing, consistency, and reproducibility. To some extent, nonspecific protein-to-fiber binding is minimized by pretreating the fiber surfaces with protein-containing blocking agents (e.g., BSA) 37 or by chemically modifying them with protein-repelling molecules (e.g., polyethylene glycol) 38 . Nonionic detergents (e.g., Tween-20 in this study), on the other hand, are commonly used to efficiently saturate nonspecific protein binding sites on nitrocellulose fiber surfaces 39 .…”

Section: Resultsmentioning

confidence: 99%

Spike- and nucleocapsid-based gold colloid assay toward the development of an adhesive bandage for rapid SARS-CoV-2 immune response detection and screening

Boumar,

Deliorman,

Sukumar

et al. 2023

Microsyst Nanoeng

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Immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies are important biomarkers used for the diagnosis and screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in both symptomatic and asymptomatic individuals. These antibodies are highly specific to the spike (S) and nucleocapsid (N) proteins of the SARS-CoV-2 virus. This paper outlines the development steps of a novel hybrid (vertical-lateral-vertical) flow assay in the form of a finger-stick point-of-care device, similar to an adhesive bandage, designed for the timely detection and screening of IgM and IgG immune responses to SARS-CoV-2 infections. The assay, comprising a vertically stacked plasma/serum separation membrane, conjugate pad, and detection (readout) zone, utilizes gold nanoparticles (AuNPs) conjugated with SARS-CoV-2 S and N proteins to effectively capture IgM and IgG antibodies from a pinprick (~15 µL) of blood in just one step and provides results of no immune IgM−/IgG−, early immune IgM+/IgG−, active immune IgM+/IgG+ or immune IgM−/IgG+ in a short amount of time (minutes). The adhesive bandage-like construction is an example of the design of rapid, low-cost, disposable, and easy-to-use tests for large-scale detection and screening in households. Furthermore, the bandage can be easily adjusted and optimized to detect different viral infections as they arise by simply selecting appropriate antigens related to pandemics and outbreaks.

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Section: Resultsmentioning

confidence: 99%

Spike- and nucleocapsid-based gold colloid assay toward the development of an adhesive bandage for rapid SARS-CoV-2 immune response detection and screening

Boumar,

Deliorman,

Sukumar

et al. 2023

Microsyst Nanoeng

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“…Zeng et al (28) have studied the effects of polyethylene glycol (PEG) on protein fixation, electrotransfer from SDS-polyacrylamide gels onto PVDF membranes and immunoblotting. Serum proteins were resolved by SDS-PAGE and the gel was then immersed in a 30% PEG 2000 buffer for 2 h for reversibly fixing the proteins (unlike trichloroacetic acidsulfosalicylic acid or acetic acid-methanol systems that irreversibly fixes proteins).…”

Section: Polyethylene Glycol Mediated Significant Enhancement Of the mentioning

confidence: 99%

Other Notable Protein Blotting Methods: A Brief Review

Kurien

Scofield

2015

Methods in Molecular Biology

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Proteins have been transferred from the gel to the membrane by a variety of methods. These include vacuum blotting, centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low- and high-molecular-weight proteins, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before matrix-assisted laser desorption/ionization tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter.

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Lane distortions in gel electrophoresis patterns

Starita‐Geribaldi¹,

Houri²,

Sudaka³

1993

Electrophoresis

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Distortions effects were studied as a function of gel environment, apparatus design and buffer type. Outward lanes distortions pronounced in low conductivity buffers for both continuous buffer systems and stacking gels, were field strength dependent. In discontinuous buffer systems, the moving boundary of the Laemmli buffer system deformed depending on the environment. In the Michov buffer system, the high conductivity resolving gel surrounded by a low conductivity electrode buffer, displayed straight pathways until a field strength of 15 V/cm, permitting to obtain information about lanes distortions depending on the acrylamide matrix structure. A decreased ammonium persulfate concentration, e.g. to 0.03%, induced phase segregation in gels at low temperature during the run: a critical endpoint near 11 degrees C for a gel in the Michov buffer system under conditions of electrophoresis was supposed. Gel collapse resulted in inward deviations in the lanes of the pattern and also faint bands in the zones where the gel was retracted. Besides this effect of catalyst concentration, the inwardly distorted pattern depended on temperature, field strength, solvent and hydrolysis due to gel aging. Amendments for overcoming lane distortions are suggested.

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Polyethylene glycol significantly enhances the transfer of membrane immunoblotting

Cited by 12 publications

References 33 publications

Spike- and nucleocapsid-based gold colloid assay toward the development of an adhesive bandage for rapid SARS-CoV-2 immune response detection and screening

Spike- and nucleocapsid-based gold colloid assay toward the development of an adhesive bandage for rapid SARS-CoV-2 immune response detection and screening

Other Notable Protein Blotting Methods: A Brief Review

Lane distortions in gel electrophoresis patterns

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