scholarly journals Enhancement of Binding Affinity of Anti-Hapten Polyclonal IgG Recognizing Mitragynine using Affinity Purification

2021 ◽  
Vol 29 (4) ◽  
Author(s):  
Radhiahtul Raehan Mustafa ◽  
Rashidah Sukor ◽  
Siti Mariam Mohd Nor ◽  
Nazamid Saari ◽  
Farina Mustaffa Kamal ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Biomedical Applications ◽  
Polyclonal Antibodies ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sds Page ◽  
Elution Peak ◽  
Polyclonal Igg ◽  
Aromatic Ether

Antibodies are glycoproteins found in peritoneal fluid, serum, and blood. The antibody-based assay has been used for broad applications such as immunodiagnostic and other biomedical applications. Depending on the intended application, a highly purified polyclonal antibody could be used as an alternative. Purification of antibodies from anti-sera has been proven as one of the methods to enhance the binding affinity of antibodies towards its antigen. We report herein the enhancement of the binding affinity of anti-hapten polyclonal IgG recognizing mitragynine using affinity purification. Serum from the terminal bleed of New Zealand White (NZW) rabbits immunized with mitragynine conjugated with cationized– bovine serum albumin at methyl ester (C22-MG-cBSA), or aromatic ether modification (C9-MG-cBSA) were subjected to HiTrap Protein G affinity purification using fast protein liquid chromatography (FPLC). The elution peak from chromatography fractions was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Here, we report the binding of polyclonal antibodies produced from inoculation of either C22-MG-cBSA or C9-MG-cBSA immunogens of which mitragynine-ovalbumin (MG-OVA) was used as coating antigen in the ELISA assay. Non purified anti-sera from C22-MG-cBSA-inoculated rabbits showed higher titer than C9-MG-cBSA at 1/128 000 and 1/32 000 dilutions, respectively. The affinity of purified poly-IgGs from rabbits immunized with C22-MG-cBSA showed a mean Kd value of 7.965 × 10-6 μM, which was lower than those immunized with C9-MG-cBSA at mean Kd of 1.390 × 10-4 μM. In addition, the purified poly- IgGs showed higher binding towards MG-OVA than non-purified anti-sera at comparable protein concentrations. These results indicated that the higher binding affinity of purified polyclonal IgG is due to the reduced competition among polyclonal antibodies with non- IgG proteins that co-existed in the non-purified anti-sera after the affinity purification.

Evaluation for Protein Binding Affinity of Maghemite and Magnetite Nanoparticles

2007 ◽  
Vol 7 (11) ◽  
pp. 3706-3708 ◽  
Author(s):  
Se Chan Kang ◽  
Yong Jun Jo ◽  
Jong Phil Bak ◽  
Ki-Chul Kim ◽  
Young-Sung Kim
Keyword(s):  
Protein Binding ◽  
Binding Affinity ◽  
Magnetite Nanoparticles ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Lung Cancer ◽  
Maghemite Nanoparticles ◽  
Binding Ability ◽  
Sds Page ◽  
Lung Cancer A549 Cell

We investigated the protein binding affinity of magnetite (Fe3O4) and maghemite (γ-Fe2O3) nanoparticles with against non-characterized protein from human lung cancer A549 cell line on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The binding ability of maghemite was 400 ng/mg. According to the SDS-PAGE results, the protein binding affinity of maghemite nanoparticles is stronger than magnetite nanoparticles. These data suggest that a protein can be detected with maghemite nanoparticles.

scholarly journals Purification and Characterisation of Lectin Isolated from Nigeria Achatina achatina Snail.

2020 ◽  
Vol 5 (3) ◽  
pp. 001-009
Author(s):  
Odiegwu C.N.C. ◽  
Ukaejiofo E.O. ◽  
Tothill I.E. ◽  
Chianella I. ◽  
Okey-Onyesolu C.F.
Keyword(s):  
Molecular Weight ◽  
Crude Extract ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Carbohydrate Binding ◽  
Protein Assay ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Specific Sugar

Lectins are carbohydrate-binding proteins that are highly specific for sugar moieties of other molecules. They perform recognition on the cellular and molecular level and play numerous roles in biological recognition phenomena involving cells, carbohydrates, and proteins. Blood groups are inherited characters which give rise to antigen-antibody reaction. A total of 120 samples of local (Nigeria) Achatina achatina snail specie were collected, authenticated at the Zoology Department of the University of Nigeria, Nsukka and 80mls of pooled crude lectin extract was obtained. Purifications were performed on 20mls of the crude extract in three steps viz, Ammonium sulphate precipitation and Dialysis (Partial purifications), Con A Sepharose 4B affinity chromatography column (Complete purification). The affinity purified lectin was used for all the actual tests conducted in this research. The crude, partially and complete/affinity purified lectin extracts were subjected to Haemagglutination tests, Protein Assay and Specific Sugar determinations. The molecular weight was assessed by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The results of the research showed as follows: On complete/affinity purification, 15mls of pure sample containing only the high molecular weight lectin was obtained. The respective haemagglutination tests on the crude, partially and affinity purified lectin showed on standardisation, preferential agglutination with Blood group A type. The Protein contents of the lectin was deduced to be as follows: The crude extract contains 13.5mg/dl, Dialysed precipitate – 5.7mg/dl, Dialysed supernatant – 5.0mg/dl and the Affinity purified Lectin – 0.422mg/dl. Galactose N-acetyl amine (Gal NAc) residue was determined to be its specific sugar. The SDS-PAGE analysis showed the molecular weight of the lectin to be 250 KDaltons. This research has therefore succeeded in the Purification, Characterisation and illustration of the lectinic properties of the local Nigeria snail - Achatina achatina.

Evaluation for Protein Binding Affinity of Maghemite and Magnetite Nanoparticles

2007 ◽  
Vol 7 (11) ◽  
pp. 3706-3708 ◽  
Author(s):  
Se Chan Kang ◽  
Yong Jun Jo ◽  
Jong Phil Bak ◽  
Ki-Chul Kim ◽  
Young-Sung Kim
Keyword(s):  
Protein Binding ◽  
Binding Affinity ◽  
Magnetite Nanoparticles ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Lung Cancer ◽  
Maghemite Nanoparticles ◽  
Binding Ability ◽  
Sds Page ◽  
Lung Cancer A549 Cell

We investigated the protein binding affinity of magnetite (Fe3O4) and maghemite (γ-Fe2O3) nanoparticles with against non-characterized protein from human lung cancer A549 cell line on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The binding ability of maghemite was 400 ng/mg. According to the SDS-PAGE results, the protein binding affinity of maghemite nanoparticles is stronger than magnetite nanoparticles. These data suggest that a protein can be detected with maghemite nanoparticles.

scholarly journals Triticum mosaic virus: A New Virus Isolated from Wheat in Kansas

Plant Disease ◽  
2008 ◽  
Vol 92 (5) ◽  
pp. 808-817 ◽  
Author(s):  
Dallas L. Seifers ◽  
T. J. Martin ◽  
Tom L. Harvey ◽  
John P. Fellers ◽  
James P. Stack ◽  
...  
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cesium Chloride ◽  
High Plains ◽  
Flight Mass Spectrometry ◽  
Sds Page ◽  
Systemic Symptoms

In 2006, a mechanically-transmissible and previously uncharacterized virus was isolated in Kansas from wheat plants with mosaic symptoms. The physiochemical properties of the virus were examined by purification on cesium chloride density gradients, electron microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), sequencing of the nucleotides and amino acids of the coat protein, and immunological reactivity. Purified preparations contained flexuous, rod-shaped particles that resembled potyviruses. The coat protein was estimated from SDS-PAGE to have a mass of approximately 35 kDa. Its amino acid sequence, as deduced from DNA sequencing of cloned, reverse-transcribed viral RNA and separately determined by time-of-flight mass spectrometry, was most closely related (49% similarity) to Sugarcane streak mosaic virus, a member of the Tritimovirus genus of the family Potyviridae. The virus gave strong positive reactions during enzyme-linked immunosorbent assays using polyclonal antibodies raised against purified preparations of the cognate virus but gave consistent negative reactions against antibodies to Wheat streak mosaic virus (WSMV), other wheat potyviruses, and the High Plains virus. When the virus was inoculated on the WSMV-resistant wheat cv. RonL, systemic symptoms appeared and plant growth was diminished significantly in contrast with WSMV-inoculated RonL. Taken together, the data support consideration of this virus as a new potyvirus, and the name Triticum mosaic virus (TriMV) is proposed.

Comparative HPLC, SDS-PAGE, and Immunoblot Analyses of Dental Enamel Proteins

1996 ◽  
Vol 10 (2) ◽  
pp. 150-158 ◽  
Author(s):  
O.H. Ryu ◽  
C.-C. Hu ◽  
J.P. Simmer
Keyword(s):  
Rna Splicing ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sequence Divergence ◽  
Dental Enamel ◽  
Reversed Phase ◽  
Enamel Matrix ◽  
Sds Page ◽  
Exon 4

The primary structures of amelogenins expressed from different genes vary because of DNA sequence divergence and variations in alternative RNA splicing. The pattern of splicing is unique for each amelogenin gene yet investigated, even when two copies of the gene are expressed in the same cell. Despite the high conservation of amelogenin sequences, diversity in the pattern of RNA splicing leads to significant differences in the number and character of amelogenin isoforms in the developing enamel matrix. Since conservation of molecular structure is an indicator of functional significance, we compared enamel protein preparations from rat, porcine, rabbit, and opossum developing tooth organs. Enamel extracts were fractionated by reversed-phase high-performance liquid chromatography (HPLC) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analyses were performed with polyclonal antibodies raised against recombinant murine amelogenin and the polypeptide encoded by murine exon 4. The opossum enamel extract produced the simplest chromatogram, suggesting that fewer proteins are secreted into the developing enamel matrix. The predominant opossum amelogenin has an apparent molecular mass of 28 kDa and reacts strongly with the recombinant amelogenin antibody but is not recognized by the murine exon 4 antibody. Opossum amelogenin mRNA was amplified with murine amelogenin primers specific for the amino- and carboxyl-terminal coding regions. The mobility of the amplification products on 4% agarose gels indicates that the leucine-rich amelogenin polypeptide (LRAP) is expressed in the opossum and that the major amelogenin is larger than its homologue in the mouse. We conclude that the alternative splicing of amelogenins pre-dates the metatherian and eutherian divergence over 100 million years ago.

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).

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>

Majra Honey Abrogated the Normal and Cancer Cells Proliferation Inhibition by Juniperus procera Extract and Extract/Honey Generated AgNPs

2020 ◽  
Vol 20 (8) ◽  
pp. 970-981
Author(s):  
Hamed A. Ghramh ◽  
Essam H. Ibrahim ◽  
Mona Kilnay
Keyword(s):  
Anticancer Activity ◽  
Cancer Cells ◽  
Biological Activities ◽  
Sugar Content ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bioactive Molecules ◽  
Sds Page ◽  
Splenic Cells ◽  
Juniperus Procera

Background: Juniperus procera and Majra honey are well-known as a folk medicine in many countries. Objectives: This work aimed to study the immunomodulatory effects after mixing Majra honey, J. procera water leaves extract and silver Nanoparticles (AgNPs) on immune or cancer cells. Methods: Juniperus procera water leaves extract and 20% Majra honey were prepared. Both the extract and honey were used separately to synthesize AgNPs. AgNPs were characterized using UV/Vis spectrophotometry and electron microscopy. Bioactive molecules in honey and the extract were explored using Fourier Transform Infrared (FT-IR) spectroscopy. Protein profile of honey was explored using Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) and honey sugar content was determined using High- Performance Liquid Chromatography (HPLC). Biological activities of honey and the extract were tested. Results: The results demonstrated the ability of the extract/honey to produce AgNPs in a spherical shape. The extract/honey contained many functional groups. SDS-PAGE of Majra honey showed many protein bands. HPLC revealed honey is of good quality and no external additives are added to it. The extract and extract+ AgNPs inhibited the growth of normal rat splenic cells while honey stimulated it. The extract+honey turned stimulatory to the splenic cells’ growth and significantly diminished the inhibitory potential of the extract containing AgNPs. Both the extract and honey have antimicrobial activities, this potential increased in the presence of AgNPs. Honey and Honey+AgNPs inhibited HepG2 cancer cell proliferation while Hela cell growth inhibited only with honey+AgNPs. Conclusion: Both honey and the extract have antibacterial and immunomodulatory potentials as well as the power to produce AgNPs. Majra honey alone showed anticancer activity against HepGe2 cells, but not against Hela cells, and when contained AgNPs had anticancer activity on both cell lines. Mixing of Majra honey with J. procera extract showed characterized immunomodulatory potentials that can be described as immunostimulant.

scholarly journals Study on cocoonase, sericin, and degumming of silk cocoon: computational and experimental

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Preeti Anand ◽  
Jay Prakash Pandey ◽  
Dev Mani Pandey
Keyword(s):  
San Francisco ◽  
Tertiary Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Evolutionary Genetics ◽  
Imaging Analysis ◽  
Silk Cocoon ◽  
Natural Color ◽  
Sds Page

Abstract Background Cocoonase is a proteolytic enzyme that helps in dissolving the silk cocoon shell and exit of silk moth. Chemicals like anhydrous Na2CO3, Marseille soap, soda, ethylene diamine and tartaric acid-based degumming of silk cocoon shell have been in practice. During this process, solubility of sericin protein increased resulting in the release of sericin from the fibroin protein of the silk. However, this process diminishes natural color and softness of the silk. Cocoonase enzyme digests the sericin protein of silk at the anterior portion of the cocoon without disturbing the silk fibroin. However, no thorough characterization of cocoonase and sericin protein as well as imaging analysis of chemical- and enzyme-treated silk sheets has been carried out so far. Therefore, present study aimed for detailed characterization of cocoonase and sericin proteins, phylogenetic analysis, secondary and tertiary structure prediction, and computational validation as well as their interaction with other proteins. Further, identification of tasar silkworm (Antheraea mylitta) pupa stage for cocoonase collection, its purification and effect on silk sheet degumming, scanning electron microscope (SEM)-based comparison of chemical- and enzyme-treated cocoon sheets, and its optical coherence tomography (OCT)-based imaging analysis have been investigated. Various computational tools like Molecular Evolutionary Genetics Analysis (MEGA) X and Figtree, Iterative Threading Assembly Refinement (I-TASSER), self-optimized predicted method with alignment (SOPMA), PROCHECK, University of California, San Francisco (UCSF) Chimera, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) were used for characterization of cocoonase and sericin proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein purification using Sephadex G 25-column, degumming of cocoon sheet using cocoonase enzyme and chemical Na2CO3, and SEM and OCT analysis of degummed cocoon sheet were performed. Results Predicted normalized B-factors of cocoonase and sericin with respect to α and β regions showed that these regions are structurally more stable in cocoonase while less stable in sericin. Conserved domain analysis revealed that B. mori cocoonase contains a trypsin-like serine protease with active site range 45 to 180 query sequences while substrate binding site from 175 to 200 query sequences. SDS-PAGE analysis of cocoonase indicated its molecular weight of 25–26 kDa. Na2CO3 treatment showed more degumming effect (i.e., cocoon sheet weight loss) as compared to degumming with cocoonase. However, cocoonase-treated silk cocoon sheet holds the natural color of tasar silk, smoothness, and luster compared with the cocoon sheet treated with Na2CO3. SEM-based analysis showed the noticeable variation on the surface of silk fiber treated with cocoonase and Na2CO3. OCT analysis also exemplified the variations in the cross-sectional view of the cocoonase and Na2CO3-treated silk sheets. Conclusions Present study enlightens on the detailed characteristics of cocoonase and sericin proteins, comparative degumming activity, and image analysis of cocoonase enzyme and Na2CO3 chemical-treated silk sheets. Obtained findings illustrated about use of cocoonase enzyme in the degumming of silk cocoon at larger scale that will be a boon to the silk industry.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

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