Isothiocyanato‐substituted hydrophilic polymer beads for immobilization of enzymes

Kitano, Hiromi; Hasegawa, Masanori; Kaku, Takashi; Ise, Norio

doi:10.1002/app.1990.070390204

Cited by 10 publications

(9 citation statements)

References 13 publications

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“…Medical diagnostics rely on immobilized proteins since most immunoassays employ immobilized antibodies or enzymes. Protein immobilization can be accomplished in several ways including adsorption onto solid supports, entrapment into membranes and films, and covalent binding to carriers such as polymeric beads. , Covalent binding can be performed in many ways; solid supports often possess hydroxy, amino, amide, and carboxy groups that must be activated before they are able to directly react with proteins. In our approach, the immobilization was done through interaction of the terminal amino group of the protein, e.g., concanavalin A or Protein G with the epoxy groups of the latex particles bound to the wall of the fused silica capillary.…”

Section: Resultsmentioning

confidence: 99%

“…A specific antibody bound to a support allows the capture and detection of low-abundance proteins. The most commonly used protein for binding monoclonal and polyclonal IgGs is protein G isolated from Streptococci bacterium . Immunoglobulin G often has to be captured and preconcentrated from solutions containing serum albumin protein.…”

Section: Resultsmentioning

confidence: 99%

“…The most commonly used protein for binding monoclonal and polyclonal IgGs is protein G isolated from Streptococci bacterium. 46 Immunoglobulin G often has to be captured and preconcentrated from solutions containing serum albumin protein. In our experiments, we used a recombinant form of protein G that lacks the albumin binding region of the native molecule, leaving two IgG binding sites that can bind all subclasses of bovine IgG.…”

Section: Enrichment Of Phosphopeptides With Iron-imac Ps-capillary β-...mentioning

confidence: 99%

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Derivatized Nanoparticle Coated Capillaries for Purification and Micro-extraction of Proteins and Peptides

Bakry¹,

Gjerde²,

Bonn³

2006

J. Proteome Res.

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Various methods are used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. One of the most powerful methods is affinity purification, also called affinity chromatography, whereby the proteins of interest are purified by virtue of their specific binding properties to an immobilized ligand. Affinity purification is becoming more widely used for exploring post-translation modifications and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. Our work was aimed to immobilize proteins or ligands for affinity purification of antibodies, fusion-tagged proteins and other proteins and peptides. Selected proteins or peptides are efficiently extracted and enriched using chemically derivatized walls of a fused silica capillary column. In this paper, we present an open tubular capillary, where the inner wall of a fused silica capillary was derivatized by covalent binding of modified polystyrene latex particles. The capillaries were derivatized with iminodiacetic acid and loaded with Fe3+ or Ni2+ for the purification and enrichment of phosphopeptides or His-tagged proteins, respectively. The latex coated capillaries have been successfully applied to enrich phosphopeptides from beta-casein tryptic digest and ovalbumin tryptic digest at a micro volume scale with recoveries ranging from 92 to 95%. The capillaries have been eluted under conditions compatible with MALDI-MS without any prior desalting step. In another approach, concanavalin A (Con A) or Protein G were immobilized on the epoxy modified latex on the inner wall of the fused silica capillary for the purification of glycoproteins and immunoglobulin, respectively. The design of the capillary and the protocols used for purification permits the direct detection of eluted proteins and peptides with gel electrophoresis or with mass spectrometry. The elution volumes are passed as discrete segments of few microliters over the inner surface of the open-tube capillary, achieving enrichment factors of more than 20-fold from starting samples.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Enrichment Of Phosphopeptides With Iron-imac Ps-capillary β-...mentioning

confidence: 99%

See 1 more Smart Citation

Derivatized Nanoparticle Coated Capillaries for Purification and Micro-extraction of Proteins and Peptides

Bakry¹,

Gjerde²,

Bonn³

2006

J. Proteome Res.

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“…8 The presence of isothiocyanate groups in the resin (NEG-12-NCS) was confirmed by the specific absorption at 2100 cm-' in the infrared spectrum using a IR440 spectrophotometer (Shimadzu, Kyoto, Japan). As a carrier of isothiocyanate groups, we also used microporous hydrophilic resins, NEG-Ill (copolymer of styrene, divinylbenzene, and glycerylmethacrylate, mean pore radius 51 A), and hydrophobic resins [poly(styrene-co-divinylbenzene)], S-X4 (exclusion limit of pores, 1400 daltons; Bio-Rad, Richmond, CA), and HP-20 (mean pore radius, 150 A; Mitsubishi Chemical Industries, Tokyo).…”

Section: Polymer Resinsmentioning

confidence: 99%

Macroporous and hydrophilic polymer resins modified with isothiocyanate groups for immobilization of enzymes

Hasegawa¹,

Kitano²,

Nishida³

et al. 1990

Biotech & Bioengineering

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Hydrophilic and macroporous polymer resins composed of glycerylmethacrylate, styrene, and divinylbenzene were quite easily modified with isothiocyanate groups using a Friedel-Crafts reaction with 3-chloropropionyl chloride and subsequent nucleophilic reaction with KSCN. Alkaline phosphatase and trypsin could be covalently bound to the isothiocyanate-carrying polymer resins, and the immobilized enzymes obtained were sufficiently active and stable due to a covalent bonding via a spacer group between the carrier resins and the enzymes and also due to a hydrophilic environment around the enzymes. A heterogeneity of the immobilized enzyme was taken into account to interpret the thermal denaturation process of the enzyme immobilized onto the resins.

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“…Hydrophilic supports also are considered important for maintaining the catalytic activity of immobilized enzymes, , but efficient immobilization of enzymes may require supports with a specifically tailored hydrophilic−hydrophobic balance . Moreover, support polarity has been shown to impact the catalytic activity − (perhaps by influencing the diffusion of substrates and products , ) and even enantioselectivity of immobilized enzymes.…”

Section: Introductionmentioning

confidence: 99%

Highly Cross-Linked Azlactone Functional Supports of Tailorable Polarity

et al. 1996

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Highly cross-linked azlactone functional supports were prepared by dispersion polymerization of 2-vinyl-4,4-dimethylazlactone (VDM), 2-hydroxyethyl methacrylate (HEMA), and trimethylolpropane trimethacrylate (TMPTMA). The supports were recovered in high yield and possessed high surface areas. Individual particles, which ranged from 10 to 300 µm in diameter, were irregularly shaped and comprised of filamentous strands. Support polarity was tailored through increases in the HEMA level in the polymer formulations and was quantified by a lipophilicity index derived from solvent partition data collected on the constituent monomers. Support reactivity, as determined by azlactone aminolysis and measured by IR, was found to increase linearly with increasing support polarity. This effect was shown to be independent of the support cross-link density.

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Isothiocyanato‐substituted hydrophilic polymer beads for immobilization of enzymes

Cited by 10 publications

References 13 publications

Derivatized Nanoparticle Coated Capillaries for Purification and Micro-extraction of Proteins and Peptides

Derivatized Nanoparticle Coated Capillaries for Purification and Micro-extraction of Proteins and Peptides

Macroporous and hydrophilic polymer resins modified with isothiocyanate groups for immobilization of enzymes

Highly Cross-Linked Azlactone Functional Supports of Tailorable Polarity

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