The NcGRA7gene encodes the immunodominant 17 kDa antigen ofNeospora caninum

Parasitology ◽  
2006 ◽  
Vol 134 (1) ◽  
pp. 41-50 ◽  
Author(s):  
G. ÁLVAREZ-GARCÍA ◽  
A. PITARCH ◽  
A. ZABALLOS ◽  
A. FERNÁNDEZ-GARCÍA ◽  
C. GIL ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Neospora Caninum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Peptide Sequencing ◽  
Dense Granule ◽  
Cdna Clones ◽  
2D Page ◽  
Amino Terminal ◽  
Proteomic Approach ◽  
Sds Page

ANeospora caninum17–19 kDa antigenic protein fraction (p17) in one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE) is the immunodominant antigen recognized by sera from bovines naturally infected byN. caninum. To identify the proteins making up the p17 fraction, we screened a newN. caninumtachyzoite cDNA library with an affinity-purified antibody against p17 (APA17). We isolated several cDNA clones with 100% sequence identity to the NcGRA7gene. This previously described gene encodes a dense granule protein with an apparent molecular mass of 33 kDa. A second line of evidence emerged through a combined proteomic approach associating two-dimensional PAGE (2D-PAGE) to Western blotting and to mass spectrometry to characterize the p17 fraction. Two acidic immunodominant but minority protein spots were recognized by APA17 and by bovine sera. These antigens of 17 and 33 kDa are respectively composed of 4 and 2 isoforms. Furthermore, p17 isolation by 2D-PAGE and peptide sequencing by tandem mass spectrometry yielded a partial sequence of 17 amino acids, which allowed the putative amino terminal region of the NcGRA7 protein to be identified unambiguously.The NcGRA7 protein, without the putative signal peptide at the NH2-terminus, was cloned and expressed inEscherichia coliand when the purified recombinant protein (rNcGRA7) was analysed by SDS-PAGE and mass spectrometry, 2 bands of 24 and 33 kDa were resolved and identified as NcGRA7. These results demonstrate that the immunodominant 17 kDa antigen ofN. caninumis encoded by the NcGRA7gene.

scholarly journals Absence of sugars in electrophoretically purified cytochrome b5 demonstrated by combined gas chromatography-mass spectrometry.

1981 ◽  
Vol 89 (3) ◽  
pp. 615-620 ◽  
Author(s):  
J Stadler
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cytochrome B5 ◽  
Sodium Dodecyl ◽  
Gas Chromatography Mass Spectrometry ◽  
Rat Liver Microsomes ◽  
Amino Terminal ◽  
Chromatography Mass Spectrometry ◽  
Sds Page

The problem of determining small but significant amounts of carbohydrates, in purified proteins, has been studied using the membrane protein, cytochrome b5. A newly developed method that involves direct gas chromatography-mass spectrometry of sugars obtained by hydrolysis of proteins purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) allows the identification and determination of small amounts of carbohydrates (e.g., 20 micrograms of glycoprotein containing a minimum of 0.1% monosaccharide), even in the presence of relatively high amounts of impurities. Application of this method to cytochrome b5 fragments obtained by tryptic digestion from rat liver microsomes and purified by combined gel filtration and ion exchange chromatography, followed by SDS PAGE, has consistently yielded values below 0.07 mol of the individual sugars and aminosugars per mole cytochrome b5. It is concluded that cytochrome b5, at least its trypsin-released major amino-terminal fragment, is not constitutively glycosylated.

scholarly journals A Cysteine-Reactive Small Photo-Crosslinker Possessing Caged-Fluorescence Properties: Binding-Site Determination of a Combinatorially-Selected Peptide by Fluorescence Imaging/Tandem Mass Spectrometry

2018 ◽  
Vol 19 (11) ◽  
pp. 3682
Author(s):  
Kazuki Yatabe ◽  
Masaru Hisada ◽  
Yudai Tabuchi ◽  
Masumi Taki
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Binding Site ◽  
Fluorescence Imaging ◽  
Polyacrylamide Gel Electrophoresis ◽  
Stokes Shift ◽  
Sodium Dodecyl ◽  
Tandem Mass ◽  
Sds Page ◽  
Sh Group

To determine the binding-site of a combinatorially-selected peptide possessing a fluoroprobe, a novel cysteine reactive small photo-crosslinker that can be excited by a conventional long-wavelength ultraviolet handlamp (365 nm) was synthesized via Suzuki coupling with three steps. The crosslinker is rationally designed, not only as a bioisostere of the fluoroprobe, but as a caged-fluorophore, and the photo-crosslinked target protein became fluorescent with a large Stokes-shift. By introducing the crosslinker to a designated sulfhydryl (SH) group of a combinatorially-selected peptide, the protein-binding site of the targeted peptide was deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/fluorescence imaging followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF-MS/MS) analysis.

Evidence that the bovine ovary secretes large amounts of monomeric inhibin α subunit and its isolation from bovine follicular fluid

1989 ◽  
Vol 2 (3) ◽  
pp. 189-200 ◽  
Author(s):  
P. G. Knight ◽  
A. J. Beard ◽  
J. H. M. Wrathall ◽  
R. J. Castillo
Keyword(s):  
Follicular Fluid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Ovarian Vein ◽  
Α Subunit ◽  
Amino Terminal ◽  
Rp Hplc ◽  
Reducing Conditions ◽  
Sds Page ◽  
Bovine Ovary

ABSTRACT Analysis of bovine follicular fluid (FF) using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with a sensitive immunoblotting procedure resolved several components that were immunoreactive with an antiserum directed against the n-terminus of the α subunit of human inhibin (hIα(1–32)). Under non-reducing conditions, three intensely stained bands having apparent Mr values of 116 000, 44 000 and 25 000 were present, whilst under reducing conditions only two intensely stained bands (Mr 43 000 and 21 000) were detected. The Mr 44 000 and 25 000 immunoreactive forms (non-reducing conditions) were also demonstrated in bovine utero-ovarian vein and peripheral venous plasma after subjecting samples (40 ml) to immunoaffinity concentration using Sepharose beads coupled to anti-hIα(1–32), SDS-PAGE and immunoblotting. The same approach revealed the presence of the smaller (Mr 25 000) form in bovine granulosa cell-conditioned culture medium (GCCM). Gel-permeation chromatography (Sephacryl S-200), immunoaffinity chromatography (Sepharose-anti-hIα(1–32)) and reversed-phase high-performance liquid chromatography (RP-HPLC; C18 and C8 columns) were employed to isolate from bFF (30 ml, 195 g protein) 750 μg protein which appeared essentially homogeneous by RP-HPLC and SDS-PAGE and had an Mr of 25 000 (non-reducing conditions)/21 000 (reducing conditions), identical to that of the immunoreactive component of lowest Mr found in bovine FF, utero-ovarian vein plasma, peripheral plasma and GCCM. The isolated material was highly immunoreactive with antisera against both hIα(1–32) and purified Mr 32 000 bovine inhibin but was devoid of biological activity when tested in a rat pituitary cell inhibin bioassay. Amino-terminal analysis revealed an amino acid sequence (residues 1–14) identical to that reported elsewhere for the α subunit (Mr 20 000/21 000) of bovine inhibin. In conclusion, the present study has revealed that the bovine ovary secretes considerable quantities of monomeric inhibin α subunit. The unexpected presence of this material in peripheral blood is likely to hinder attempts to obtain physiologically relevant data on circulating levels of inhibin in cattle using conventional radioimmunoassays.

scholarly journals A Proteomic Approach to Identify Zein Proteins upon Eco-Friendly Ultrasound-Based Extraction

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1838
Author(s):  
Laura Darie-Ion ◽  
Madhuri Jayathirtha ◽  
Gabriela Elena Hitruc ◽  
Marius-Mihai Zaharia ◽  
Robert Vasile Gradinaru ◽  
...  
Keyword(s):  
Protein Extraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Industrial Applications ◽  
Experimental Conditions ◽  
Proteomic Approach ◽  
Sds Page ◽  
Pharmaceutical Industries

Zein is a type of prolamin storage protein that has a variety of biomedical and industrial applications. Due to the considerable genetic variability and polyploidity of the starting material, as well as the extraction methods used, the characterization of the protein composition of zein requires a combination of different analytical processes. Therefore, we combined modern analytical methods such as mass spectrometry (MS), Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), atomic force microscopy (AFM), or Fourier transform infrared spectroscopy–attenuated total reflectance (FTIR-ATR) for a better characterization of the extracted zein. In this study, we present an enhanced eco-friendly extraction method, including grinding and sieving corn seeds, for prolamins proteins using an ultrasonic extraction methodology. The use of an ultrasonic homogenizer, 65% ethanol extraction buffer, and 710 µm maize granulation yielded the highest protein extraction from all experimental conditions we employed. An SDS PAGE analysis of the extracted zein protein mainly revealed two intense bands of approximatively 20 and 23 kDa, suggesting that the extracted zein was mostly α-zein monomer. Additionally, MS analysis revealed as a main component the α-zein PMS2 (Uniprot accession no. P24450) type protein in the maize flour extract. Moreover, AFM studies show that extracting zein with a 65% ethanol and a 710 µm granulation yields a homogeneous content that could allow these proteins to be employed in future medical applications. This research leads to a better understanding of zeins content critical for developing new applications of zein in food and pharmaceutical industries, such as biocompatible medical vehicles based on polyplexes complex nanoparticles of zein with antimicrobial or drug delivery properties.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

Identifikasi Daging Tikus Pada Produk Baso Dengan Metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

2018 ◽  
Vol 26 (2) ◽  
pp. 058
Author(s):  
Anna P. Roswiem ◽  
Triayu Septiani
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sds Page

<em>Bahan<strong> </strong>baku untuk membuat baso adalah daging hewan, pada umumnya dari daging sapi, ayam, ikan dan babi. Di beberapa daerah di Indonesia terjadi kasus baso tikus. Tujuan penelitian ini adalah menguji ada tidaknya kandungan daging tikus pada produk baso yang dijual di pasar Cempaka Putih-Kecamatan Kramat Jakarta Pusat dan di pedagang baso atau mie baso di sekitar kampus Universitas YARSI Jakarta. Daging adalah protein salah satu metode untuk mengidentifikasi protein adalah metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE).<strong> </strong>Hasil penelitian menunjukkan bahwa dari 6 sampel baso terindikasi ada 2 sampel baso dengan nomor 1 dan 5 yang dibuat dari campuran daging sapi dan tikus; ada 1 sampel baso dengan nomor 6 yang terbuat dari daging tikus; dan 2 sampel baso dengan nomor 2 dan 3 yang terbuat dari campuran sapi  dan babi, dan hanya 1 sampel baso dengan nomor sampel 4 yang benar-benar terbuat dari daging sapi.</em>

Characterization of Major Allergens of Local Mud Crab (Scylla serrata)

2015 ◽  
Vol 12 (1) ◽  
pp. 1 ◽  
Author(s):  
Rosmilah Misnan ◽  
Nurul Izzah Abdul Rahman ◽  
Zailatul Hani Mohd Yadzir ◽  
Noormalin Abdullah ◽  
Mohd Faizal Bakhtiar ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Arginine Kinase ◽  
Mass Spectrometry Analysis ◽  
Scylla Serrata ◽  
Mud Crab ◽  
Meat Allergy ◽  
Proteomic Approach ◽  
The World ◽  
Major Allergens ◽  
Crab Meat

Crab meat is widely consumed in several countries around the world. However, when consumed, crab meats are frequent cause of allergic reactions throughout the world. Scylla serrata is among the most common mud crab in Malaysia. In a previous study two major allergens of mud crab at 36 and 41 kDa was identified. Thus, the aim of this study is to further identify these major allergens by a proteomic approach. Protein extract was prepared and resolved by 2-dimensional electrophoresis (2-DE). Immunoblotting was then performed using reactive sera from patients with crab allergy. Major allergenic spots were then excised from the 2-DE gel and analysed by mass spectrometry. The 2-DE profile of the extract revealed approximately >100 protein spots between pH of 4.00 to 8.00. Mass spectrometry analysis has identified the 36 and 41 kDa proteins as tropomyosin and arginine kinase, respectively. Our findings indicated that tropomyosin and arginine kinase play a major role in allergic reaction to mud crab meat among local patients with crab meat allergy, and should be included in diagnostics and therapeutic strategies of this allergy.

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