Lipopolysaccharide aggregates in native agarose gels detected by reversible negative staining with imidazole and zinc salts

Rodríguez, Cynthia; Hardy, Eugenio

doi:10.1016/j.ab.2015.06.020

Cited by 7 publications

(10 citation statements)

References 17 publications

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“…Using lysozyme as a model protein, we have found that imidazolezinc may be combined with Coomassie brilliant blue R-250 into a double-staining process to enable the use of NAGE for detecting and studying protein-LPS interactions ( Figure 1B) [37]. Specifically, our results validate that lysozyme can disrupt LPS aggregates and that concentration of either LPS or LZM has a significant effect on the amount of LPS disaggregated and the amount of LZM-LPS complexes formed.…”

Section: Interaction Of Proteins With Lps Aggregatessupporting

confidence: 58%

“…Very recently, we have reported the reversible negative staining with imidazole and zinc salts of LPS aggregates separated in agarose gels [37]. After incubating the electrophoresed agarose gel in 0.2 M imidazole solution, reagents (e.g., sulfate groups, Tris) that can potentially hamper the reverse staining of LPS aggregates are washed out of the gel matrix [38].…”

Section: Separation and Detection Of Lps Aggregatesmentioning

confidence: 99%

“…The benefit of using NAGE combined with the double-staining detection is that it neither alters the LPS aggregates structurally nor disrupts the native state of the proteins and LPS. Furthermore, the NAGE method can resolve LPS complexes from free cationic proteins [37].…”

Section: Interaction Of Proteins With Lps Aggregatesmentioning

confidence: 99%

“…The outer core polysaccharide is linked to a core oligosaccharide and lipid A. B) Detection of lysozyme-LPS interaction and complex formation by double staining with imidazole-zinc/Coomassie brilliant blue R-250 [37]. C) Schematic representation of slab PAGE-mass spectrometry [56,57].…”

Section: Introductionmentioning

confidence: 99%

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Lipopolysaccharide (LPS) and Protein-LPS complexes: Detection and Characterization by Gel Electrophoresis, Mass Spectrometry and Bioassays

Hardy¹,

Rodríguez²

2016

Biol Med (Aligarh)

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A prerequisite to the discovery and characterization of lipopolysaccharide (LPS) interaction with specific receptors and the resulting pathophysiological effects is the comprehensive structural analysis of LPS species. This brief review is aimed to summarize the use of gel electrophoresis linked to other biochemical technologies for detecting and characterizing LPSs. Lipopolysaccharide aggregates alone or mixtures containing LPSs and proteins/peptides can be separated by native agarose gel electrophoresis (NAGE), after which LPSs are detected with imidazole and zinc salts. A double-staining process with Coomassie brilliant blue R-250 enables the use of NAGE for detecting and studying protein-LPS interactions. For compositional analysis, the LPS aggregates are separated, with high resolution, by surfactant-polyacrylamide gel electrophoresis. After reverse staining with zinc-imidazole and elution from gel microparticles, glycoform-specific LPSs are ready for structural and biological analysis. For sequence analysis based on tandem electrospray ionization mass spectrometry (ESI-MS/MS) of oligosaccharides, LPSs are subjected to mild acid hydrolysis, dephosphorylation and permethylation. Also, O-deacylated LPS forms can be analyzed by matrix-assisted laser desorption/ionization-time of flight-MS. By comparison to spectra of unpurified LPSs, mass spectra of the micropurified LPSs show reduce heterogeneity and increased signal-to-noise ratios. Furthermore, the micropurification of LPSs prior to MS allows a higher sensitivity of detection for less abundant LPS glycoforms. The micropurified LPS fractions can be used to form self-assembled nanoaggregates which may be detected by dynamic light scattering. The effect of the O-side chain length on the Z-potential of LPS aggregates may be estimated by measurements based on laser Doppler electrophoresis. The thus obtained glycoform-specific LPSs are not only intact chemically but also biologically active as tested by e.g. Limulus amoebocyte lysate test, TNF-α assay and agonistic effect on human Toll-like receptor 4.

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Section: Interaction Of Proteins With Lps Aggregatessupporting

confidence: 58%

Section: Separation and Detection Of Lps Aggregatesmentioning

confidence: 99%

Section: Interaction Of Proteins With Lps Aggregatesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Lipopolysaccharide (LPS) and Protein-LPS complexes: Detection and Characterization by Gel Electrophoresis, Mass Spectrometry and Bioassays

Hardy¹,

Rodríguez²

2016

Biol Med (Aligarh)

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“…Recombinant protein expression was assessed after 0, 1, 1.5, 2, 2.5, 3, and 4 h of induction. Proteins were stained with Coomassie blue R-250 and separated by SDS-polyacrylamide gel electrophoresis (PAGE; Cheng et al, 2002;Choveaux et al, 2012;Rodríguez and Hardy, 2015).…”

Section: Expression Vector Construction and Overexpression Of Recombimentioning

confidence: 99%

Molecular cloning, overexpression, purification, and sequence analysis of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide

Hou

Ding

et al. 2016

Genet. Mol. Res.

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ABSTRACT. The complementary DNA (cDNA) of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide (FTL) gene was successfully cloned using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing FTL cDNA and overexpressed it in Escherichia coli using pET28a plasmids. The expressed protein was then purified by nickel chelate affinity chromatography. The cloned cDNA fragment was 580 bp long and contained an open reading frame of 525 bp. The deduced protein sequence was composed of 175 amino acids and had an estimated molecular weight of 19.90 kDa, with an isoelectric point of 5.53. Topology prediction revealed one N-glycosylation site, two casein kinase II phosphorylation sites, one N-myristoylation site, two protein kinase C phosphorylation sites, and one cell attachment sequence. Alignment indicated that the nucleotide and deduced amino acid sequences are highly conserved across several mammals, including Homo sapiens, Cavia porcellus, Equus caballus, and Felis catus, among others. The FTL gene was readily expressed in E. coli, which gave rise to the accumulation of a polypeptide of the expected size (25.50 kDa, including an N-terminal polyhistidine tag).

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Analysis of the degradation of amyloid beta fibrils after separation via the combination of non‐denaturing agarose electrophoresis and Congo red dye staining

Kawano

Nakanishi

Zako

et al. 2019

Separation Science Plus

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A combinational method for the separation and the degradation of amyloid‐β fibrils in the biological samples is developed to suppress the amyloid‐β fibrils content. The complexes of the Congo red dye and amyloid‐β fibrils migrate to the anode in a non‐denaturing agarose electrophoresis owing to its negative charge, thus separating the complex containing amyloid‐β fibrils from the human plasma proteins and the amyloid‐β monomer. The separated amyloid‐β fibrils exhibited birefringence under a polarizing microscope and immunoreactivity to the anti‐amyloid‐β antibody. The digestion of 0.23 nmol of the amyloid‐β fibrils using nattokinase at 37°C for 24 h increased with the amount of enzyme up to 9 pmol, beyond which the digestion was independent of the enzyme concentration. The digestion of 0.23 nmol of the amyloid‐β fibrils with 22.5 pmol of nattokinase at 37°C increased with time up to 6 h, following which, it became independent of time. The extraction of the separated amyloid‐β fibrils from the agarose gel accompanied the removal of major plasma proteins from the spiked plasma and amyloid‐β fibrils. The combination of the non‐denaturing agarose gel electrophoresis and the dye staining would be applicable to examine formation and degradation processes of amyloid‐β fibrils after their separation from the biological samples.

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Lipopolysaccharide aggregates in native agarose gels detected by reversible negative staining with imidazole and zinc salts

Cited by 7 publications

References 17 publications

Lipopolysaccharide (LPS) and Protein-LPS complexes: Detection and Characterization by Gel Electrophoresis, Mass Spectrometry and Bioassays

Lipopolysaccharide (LPS) and Protein-LPS complexes: Detection and Characterization by Gel Electrophoresis, Mass Spectrometry and Bioassays

Molecular cloning, overexpression, purification, and sequence analysis of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide

Analysis of the degradation of amyloid beta fibrils after separation via the combination of non‐denaturing agarose electrophoresis and Congo red dye staining

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