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Ovalbumin, native, NO X w_denatured, Clone 099_01, Mab anti_Chicken; ELISA

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[#HYB099-01] Ovalbumin, native, NO X w_denatured, Clone 099_01, Mab anti_Chicken; ELISA


HYB099-01 | Ovalbumin, native, NO X w_denatured, Clone 099_01, Mab anti_Chicken; ELISA, 200 µg.
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(1) Structural change of ovalbumin-related protein X by alkali treatment.[TOP]

Pubmed ID :29462487
Publication Date : //
Chicken egg white protein ovalbumin (OVA) undergoes a conversion to a more thermostable form by alkali treatment, which is assumed to be involved in the physiological functions of OVA. Ovalbumin-related protein X (OVAX), a chicken egg white protein, has 77% sequence similarity to OVA and binds to heparin. In this study, structure characteristics and heparin-binding affinity of alkali-treated OVAX were investigated. Cation-exchange chromatography using SP Sepharose resin showed that alkali treatment (pH 10, 55°C) of OVAX induces the occurrence of a distinct OVAX form with a less positive-charge (acidic OVAX). Circular dichroism and tryptophan-fluorescence analyses showed that the newly-formed acidic OVAX form has an 8% lower α-helical content than its native counterpart, while there is no significant difference in steric environments around tryptophan residues between the 2 forms. The OVAX structure built by homology-modeling showed that OVAX possess a basic cluster domain with α-helix equivalent to 7% of total secondary structures, which does not contain any tryptophan residues. These results suggest that, during alkali treatment, OVAX undergoes mainly a conformational change of the α-helical basic cluster domain and thereby forms acidic OVAX. Acidic OVAX induced by alkali treatment exhibited weaker interactions with Heparin Sepharose resin than native OVAX did. Our results suggest that OVAX basic cluster domain is likely a specific binding site of heparin. Consequently, it is suggested that alkali treatment causes the collapse of the OVAX heparin binding site, which might participate in regulating the functions of heparin.

Authors : Akazawa Takashi, Ogawa Masahiro, Hayakawa Shigeru, Hirata Misato, Niwa Takahiro,

(2) Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells.[TOP]

Pubmed ID :26999763
Publication Date : //
Antigen uptake by dendritic cells and intracellular routing of antigens to specific compartments is regulated by C-type lectin receptors that recognize glycan structures. We show that the modification of Ovalbumin (OVA) with the glycan-structure Lewis(X) (Le(X)) re-directs OVA to the C-type lectin receptor MGL1. Le(X)-modification of OVA favored Th1 skewing of CD4(+) T cells and enhanced cross-priming of CD8(+) T cells. While cross-presentation of native OVA requires high antigen dose and TLR stimuli, Le(X) modification reduces the required amount 100-fold and obviates its dependence on TLR signaling. The OVA-Le(X)-induced enhancement of T cell cross-priming is MGL1-dependent as shown by reduced CD8(+) effector T cell frequencies in MGL1-deficient mice. Moreover, MGL1-mediated cross-presentation of OVA-Le(X) neither required TAP-transporters nor Cathepsin-S and was still observed after prolonged intracellular storage of antigen in Rab11(+)LAMP1(+) compartments. We conclude that controlled neo-glycosylation of antigens can crucially influence intracellular routing of antigens, the nature and strength of immune responses and should be considered for optimizing current vaccination strategies.

Authors : Streng-Ouwehand Ingeborg, Ho Nataschja I, Litjens Manja, Kalay Hakan, Boks Martine Annemarie, Cornelissen Lenneke A M, Kaur Singh Satwinder, Saeland Eirikur, Garcia-Vallejo Juan J, Ossendorp Ferry A, Unger Wendy W J, van Kooyk Yvette,

(3) Transition of serine residues to the D-form during the conversion of ovalbumin into heat stable S-ovalbumin.[TOP]

Pubmed ID :25982752
Publication Date : //
Ovalbumin, a major protein in chicken egg white, is converted into a more thermostable molecular form, known as S-ovalbumin, during the storage of shell eggs. Our previous X-ray crystallographic study indicated that S-ovalbumin contains three D-Ser residues (S164, S236, and S320), which may account for its thermostability. Here, we confirmed the presence of these D-Ser residues in ovalbumin using a technique combining deuterium labeling of α-protons of amino acids and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ovalbumin from chicken egg white and recombinant ovalbumin were incubated for approximately 12 days at pH 9.5 and 37°C. They were then hydrolyzed in DCl/D2O vapor, derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and analyzed by LC-MS/MS. A time-dependent increase in the D-Ser contents in native ovalbumin was observed over a period of 7 days, reaching approximately 8%. This corresponds to a value of three serine residues per molecule, and is consistent with the prediction based on our previous crystallographic analysis. Nearly identical results were obtained with recombinant ovalbumin. We then used this technique to investigate whether D-amino acid residues could arise within other proteins under mild alkaline conditions and detected small but significant amounts of D-Ala and/or D-Ser residues that increased in a time-dependent manner in some proteins.

Authors : Miyamoto Tetsuya, Takahashi Nobuyuki, Sekine Masae, Ogawa Tetsuhiro, Hidaka Makoto, Homma Hiroshi, Masaki Haruhiko,

(4) Site-specific derivatization of avidin using microbial transglutaminase.[TOP]

Pubmed ID :24517223
Publication Date : //
Avidin conjugates have several important applications in biotechnology and medicine. In this work, we investigated the possibility to produce site-specific derivatives of avidin using microbial transglutaminase (TGase). TGase allows the modification of proteins at the level of Gln or Lys residues using as substrate an alkyl-amine or a Gln-mimicking moiety, respectively. The reaction is site-specific, since Gln and Lys derivatization occurs preferentially at residues embedded in flexible regions of protein substrates. An analysis of the X-ray structure of avidin allowed us to predict Gln126 and Lys127 as potential sites of TGase's attack, because these residues are located in the flexible/unfolded C-terminal region of the protein. Surprisingly, incubation of avidin with TGase in the presence of alkylamine containing substrates (dansylcadaverine, 5-hydroxytryptamine) revealed a very low level of derivatization of the Gln126 residue. Analysis of the TGase reaction on synthetic peptide analogues of the C-terminal portion of avidin indicated that the lack of reactivity of Gln126 was likely due to the fact that this residue is proximal to negatively charged carboxylate groups, thus hampering the interaction of the substrate at the negatively charged active site of TGase. On the other hand, incubation of avidin with TGase in the presence of carbobenzoxy-l-glutaminyl-glycine in order to derivatize Lys residue(s) resulted in a clean and high yield production of an avidin derivative, retaining the biotin binding properties and the quaternary structure of the native protein. Proteolytic digestion of the modified protein, followed by mass spectrometry, allowed us to identify Lys127 as the major site of reaction, together with a minor modification of Lys58. By using TGase, avidin was also conjugated via a Lys-Gln isopeptide bond to a protein containing a single reactive Gln residue, namely, Gln126 of granulocyte-macrophage colony-stimulating factor. TGase can thus be exploited for the site-specific derivatization of avidin with small molecules or proteins.

Authors : Spolaore Barbara, Damiano Nunzio, Raboni Samanta, Fontana Angelo,

(5) Zebavidin--an avidin-like protein from zebrafish.[TOP]

Pubmed ID :24204770
Publication Date : //
The avidin protein family members are well known for their high affinity towards D-biotin and high structural stability. These properties make avidins valuable tools for a wide range of biotechnology applications. We have identified a new member of the avidin family in the zebrafish (Danio rerio) genome, hereafter called zebavidin. The protein is highly expressed in the gonads of both male and female zebrafish and in the gills of male fish, but our data suggest that zebavidin is not crucial for the developing embryo. Biophysical and structural characterisation of zebavidin revealed distinct properties not found in any previously characterised avidins. Gel filtration chromatography and native mass spectrometry suggest that the protein forms dimers in the absence of biotin at low ionic strength, but assembles into tetramers upon binding biotin. Ligand binding was analysed using radioactive and fluorescently labelled biotin and isothermal titration calorimetry. Moreover, the crystal structure of zebavidin in complex with biotin was solved at 2.4 Å resolution and unveiled unique ligand binding and subunit interface architectures; the atomic-level details support our physicochemical observations.

Authors : Taskinen Barbara, Zmurko Joanna, Ojanen Markus, Kukkurainen Sampo, Parthiban Marimuthu, Määttä Juha A E, Leppiniemi Jenni, Jänis Janne, Parikka Mataleena, Turpeinen Hannu, Rämet Mika, Pesu Marko, Johnson Mark S, Kulomaa Markku S, Airenne Tomi T, Hytönen Vesa P,

(6) Vaccination with major outer membrane protein proteosomes elicits protection in mice against a Chlamydia respiratory challenge.[TOP]

Pubmed ID :23999313
Publication Date : //
Vaccines formulated with the Chlamydia muridarum native major outer membrane protein (nMOMP) have so far been shown to elicit the most robust protection against this pathogen. nMOMP is a membrane protein and therefore, detergents are used to keep it in solution. Detergents however, have toxic effects. To address this limitation, we tested a nMOMP proteosome vaccine and compared its ability to elicit protection against nMOMP solubilized in the detergent Z3-14. The two preparations were formulated with or without CpG + Montanide (C/M). As a control antigen we used ovalbumin. Mice vaccinated with nMOMP developed strong humoral and cell mediated Chlamydia-specific immune responses. Based on the IgG2a/IgG1 levels in serum and amounts of IFN-γ in splenocytes supernatants the immune responses were predominantly Th1-biased. The animals were subsequently challenged intranasally with 2 × 10(3)Chlamydia inclusion forming units (IFU) and the course of the infection was followed for 10 days when the mice were euthanized. Based on changes in body weight, weight of the lungs and number of IFU recovered from the lungs, mice immunized with nMOMP-Ps and nMOMP + Z3-14 adjuvanted with C/M showed the most robust protection. In summary, nMOMP-Ps should be considered as Chlamydia vaccine candidates.

Authors : Tifrea Delia F, Pal Sukumar, Toussi Deana N, Massari Paola, de la Maza Luis M,

(7) Rational development of caged-biotin protein-labeling agents and some applications in live cells.[TOP]

Pubmed ID :22035795
Publication Date : //
Biotin-(strept)avidin complex is widely used in biotechnology because of its extremely high binding constant, but there is no report describing spatiotemporally controlled formation of the complex in live cells. Here, based on X-ray crystal structure analysis and calorimetric data, we designed and synthesized photoreleasable biotins, which show greatly reduced affinity for (strept)avidin, but recover native affinity after UV irradiation. For application at the cell surface, we introduced an amine-reactive moiety into these "caged" biotin molecules. Specific fluorescence imaging of live cells that had been labeled with these agents and then UV-irradiated, was accomplished by addition of streptavidin conjugated with a fluorophore. We also demonstrated the applicability of these compounds for UV-irradiated-cell-specific drug delivery by using caged-biotin-labeled cells, a prodrug, and streptavidin conjugated with a prodrug-activating enzyme.

Authors : Terai Takuya, Maki Eri, Sugiyama Shigeru, Takahashi Yoshinori, Matsumura Hiroyoshi, Mori Yusuke, Nagano Tetsuo,

(8) Protein substitution affects glass transition temperature and thermal stability.[TOP]

Pubmed ID :20707306
Publication Date : //
When proteins are removed from their native state they suffer from two deficiencies: (1) glassy behavior with glass transition temperatures (Tg) well above room temperature and (2) thermal instability. The glassy behavior originates in multiple hydrogen bonds between amino acids on adjacent protein molecules. Proteins, like most biopolymers, are thermally unstable. Substituting ovalbumin with linear and cyclic substituents using a facile nucleophilic addition reaction can affect Tg and thermal stability. More hydrophobic linear substituents lowered Tg by interrupting intermolecular interactions and increasing free volume. More hydrophilic and cyclic substituents increased thermal stability by increasing intermolecular interactions. In some cases, substituents instituted cross-linking between protein chains that enhanced thermal stability. Internal plasticization using covalent substitution and external plasticization using low molecular weight polar liquids show the same protein structural changes and a signature of plasticization is identified.

Authors : Budhavaram Naresh K, Miller Jonathan A, Shen Ying, Barone Justin R,

(9) Targeting glycan modified OVA to murine DC-SIGN transgenic dendritic cells enhances MHC class I and II presentation.[TOP]

Pubmed ID :19818504
Publication Date : //
Dendritic cells have gained much interest in the field of anti-cancer vaccine development because of their central function in immune regulation. One of the receptors that facilitate DC-specific targeting of antigens is the DC-specific C-type lectin DC-SIGN. Although DC-SIGN is specifically expressed on human DCs, its murine homologue is not present on any murine DC subsets, which makes in vivo evaluation of potential DC-SIGN targeting vaccines very difficult. Here we describe the use of DC-SIGN transgenic mice, as a good model system to evaluate DC-SIGN targeting vaccines. We demonstrate that glycan modification of OVA with DC-SIGN targeting glycans, targets antigen specifically to bone marrow (BM)** derived DCs and splenic DCs. Glycan modification of OVA with Lewis X or Lewis B oligosaccharides, that target DC-SIGN transgenic DCs, resulted in efficient 10-fold induction of OT-II compared to unmodified OVA. Interestingly, glycan modified OVA proteins were significantly cross-presented to OT-I T cells by wild type DC, 10-fold more than native OVA, and the expression of DC-SIGN further enhanced this cross-presentation. Targeting of glycosylated OVA was neither accompanied with any DC maturation, nor the production of inflammatory or anti-inflammatory cytokines. Thus, we conclude that glycan modification of antigens and targeting to DC-SIGN enhance both CD4 and CD8 T cell responses. Furthermore, our data demonstrate that DC-SIGN transgenic mice are valuable tool for optimisation and efficiency testing of DC vaccination strategies that are designed to target in particular the human DC-SIGN receptor.

Authors : Singh Satwinder Kaur, Stephani Johannes, Schaefer Martin, Kalay Hakan, García-Vallejo Juan J, den Haan Joke, Saeland Eirikur, Sparwasser Tim, van Kooyk Yvette,

(10) Nitroavidin as a ligand for the surface capture and release of biotinylated proteins.[TOP]

Pubmed ID :19551994
Publication Date : //
Discussed here is the preparation, detailed purification, and evaluation of nitroavidin as a ligand for surface capture and release of biotinylated proteins. Avidin from chicken egg white was nitrated using dilute tetranitromethane solutions. UV-vis spectroscopy was used to show decreased binding of the biotin analogue, 2-(4'-hydroxyazobenzene)benzoic acid, HABA, to nitroavidin compared to binding of HABA to native avidin. From enzyme-linked immunosorbent assay (ELISA)-based assays of the modified avidin, it was found that there are approximately three tyrosine residues converted to nitrotyrosine out of the total four tyrosine residues in the protein tetramer. For the first time, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to demonstrate the point of nitration in nitroavidin as that of the tyrosine associated with the binding of biotin (Y33). From surface plasmon resonance spectroscopy (SPR) experiments, it is shown that biotin has less binding propensity to immobilized nitroavidin (K(D) = 4.4 +/- 1.9 x 10(-6) M) than immobilized avidin (K(D) < or = 10(-11) M). Importantly, the use of pH 10 carbonate buffer as eluent resulted in facile release of bound biotin from the nitroavidin-functionalized surfaces, allowing for readily regenerated biotin capture surfaces (reversible binding surfaces). These outcomes are important for the development of protein concentration methods directed at isolation of select proteins from a large population using gentle target protein isolation/release conditions.

Authors : Bolivar Jowell G, Soper Steven A, McCarley Robin L,