Silencing of Nrf genes in the human placenta as measured by SDS-PAGE and Western Blotting techniques

Purpose. Nitrotyrosine was quantitated in rabbit bladder muscle and mucosa using two analytical systems: Western blotting analyses and a 96-well plate quantitative analysis kit. Materials and Methods. Rabbit bladder muscle and mucosa were obtained from control rabbits. For the Western analysis, the samples were loaded into a SDS page gel and then transferred to a PVDF membrane. The optical density was measured using a Kodak Scanner. Using the 96-well plate, the samples and standards were loaded, incubated with primary and secondary antibody, washed and vacuumed with 10x wash buffer three times between each incubation period. Stop buffer was added to the plate and the results were quantified via the plate reader. Results. For both muscle and mucosa tissue, the optical density readings were linear with tissue concentration; the concentration of nitrotyrosine in the mucosa was significantly higher than in the muscle. However, whereas the Western blot analysis is based on relative optical densities, the 96-well plate kit provides a truly quantitative analysis. Discussion. Mucosa tissue displayed a higher density of nitrotyrosine than did detrusor muscle tissue. This may well be due to the significantly higher metabolic activity of the mucosa compared to the muscle.

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Generating and characterizing the anti-human CD45 monoclonal antibody

Science and Technology Development Journal ◽

10.32508/stdj.v23i3.2409 ◽

2020 ◽

Vol 23 (3) ◽

pp. 665-672

Author(s):

Giang Huong Ta ◽

Huy Quoc Nguyen ◽

Quan Dang Nguyen

Keyword(s):

Monoclonal Antibody ◽

Western Blotting ◽

Jurkat Cells ◽

Spleen Cells ◽

Myeloma Cells ◽

E Coli ◽

Common Marker ◽

Sds Page ◽

Hybridoma Technology ◽

Mab Production

Introduction: CD45 is a common marker of leukocytes. Anti-human CD45 monoclonal antibody (MAb) has been used widely in diagnosing and monitoring hematologic diseases. The aim of this study was to generate an anti-human CD45 MAb, which can be used in research and diagnosis. Methods: Recombinant human CD45RO antigen was expressed from E. coli BL21 (DE3), purified and analyzed by SDS-PAGE and Western blotting. The purified CD45RO antigen was used to immunize Balb/c mice. Spleen cells from immunized mouse were collected and fused with P3X63Ag8.653 myeloma cells to form hybridoma. Anti-CD45 antibody-secreting capacity of hybridoma clones was evaluated by ELISA assay. Anti-CD45 MAb from the culture supernatant of the chosen hybridoma clone was purified by affinity chromatography. The MAb was characterized the biochemical characteristics and biological activity. Results: Recombinant human CD45RO antigen was expressed and purified from E.coli BL21 (DE3). Injection of purified CD45RO antigen provoked the immune response in Balb/c mice. Hybridoma clones were generated successfully by the fusion of spleen cells from the selected immunized-mouse and myeloma cells. Among these hybridoma clones, one with the highest yield of MAb production was identified. The isotype of the anti-CD45 MAb created in this work is IgG2b, while its the light chain is kappa (k) type. The affinity of this MAb with CD45RO antigen is high with Kd value at the picomolar level. The anti-CD45 MAb can interact with CD45 naturally expressed on the surface of Jurkat cells in Western blotting and fluorescent immuno-staining assay. Conclusion: We have developed successfully an anti-human CD45 MAb using hybridoma technology, which can recognize CD45 in ELISA, Western blotting, and fluorescent immuno-staining analysis. Although further investigations are necessary, obviously, our anti-human CD45 MAb is potential for research and diagnosis applications.

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PURIFICATION AND CHARACTERIZATION OF REACTIVE AND NON-REACTIVE PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) FROM HUMAN PLACENTA

10.1055/s-0038-1642806 ◽

1987 ◽

Author(s):

M Philips ◽

A G Juul ◽

S Thorsen ◽

J Selmer ◽

L Thim

Keyword(s):

Monoclonal Antibody ◽

Plasminogen Activator ◽

Human Placenta ◽

Solid Phase ◽

Plasminogen Activator Inhibitor ◽

Tissue Type ◽

Single Chain ◽

Plasminogen Activator Inhibitor 1 ◽

Sds Page ◽

Pai 1

Reactive and non-reactive forms of PAI-1 have been identified in various biological materials. The structural differences between these forms remain to be determined.A monoclonal antibody specific for a non-reactive PAI-1 and a monoclonal antibody reacting with both the reactive and nonreactive form of the inhibitor were obtained by immunization with a tissue-type plasminogen activator (t-PA)-PAI-1 complex (Philips et al., Thromb Haemostas 1986; 55:213-7). These antibodies were used for the isolation of reactive and non-reactive PAI-1 by solid-phase immunoadsorption from extracts of human placenta. The inhibitor preparations were further purified by HPLC. Reactive and non-reactive PAI-1 both migrated with a Mr ∼ 52,000 when analyzed by SDS-PAGE. Furthermore, the two inhibitor forms were indistinguishable by N-terminal sequence analysis. Two N-terminal sequences were found in about equal ammounts for both the reactive and non-reactive PAI-1. They were Ser-Ala-Val-His-His-Pro-Pro- and a two residues shorter sequence (Val-His-His-Pro-Pro-). These sequences are in agreement with the published cDNA sequence of PAI-1 and shows that the inhibitor is N-terminally heterogeneously processed. The second order rate constant (ki) for the reaction between reactive PAI-1 and single-chain t-PA was about 6 106 M-1s-1. Treatment with 4 M guanidinium-HCl partially converted the non-reactive PAI-1 to a reactive form exhibiting a similar k1 for inhibition of single-chain t-PA. SDS-PAGE showed that the t-PA-PAI-1 complex could be dissociated by 1,5 M NH4OH/ 39 mM SDS resulting in the release of a PAI-1 with approximately the same Mr as native PAI-1. This indicates either that t-PA does not cleave the inhibitor or that it cleaves a peptide bond close to the C-terminus.In conclusion a non-reactive and a reactive form of PAI-1 can be purified from placenta. The two forms are distinguishable by monoclonal antibodies but they show similar Mr′ls and the same N-terminal sequences.

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Vírus do mosaico severo do caupi-CPSMV como molécula carreadora para a p28 do vírus da artrite-encefalite caprina-CAEV

Ciência Rural ◽

10.1590/s0103-84782005000600021 ◽

2005 ◽

Vol 35 (6) ◽

pp. 1363-1367 ◽

Cited By ~ 2

Author(s):

Francisco Jarbas Santos de Sousa ◽

Marcelo Róseo de Oliveira ◽

Ney de Carvalho Almeida ◽

Marlos Gomes Martins ◽

Maria Erivalda Farias de Aragão ◽

...

Keyword(s):

Western Blotting ◽

Sds Page

O vírus da Artrite-encefalite caprina (CAEV) pertence à família Retroviridae, gênero Lentivirus. O CAEV infecta caprinos do mundo inteiro causando artrite, encefalite, mamite, pneumonia e emagrecimento progressivo. Este trabalho mostra a formação de uma quimera construída através da mistura da p28 do CAEV com glutaraldeído e CPSMV, purificada por meio de cromatografia em biogel e sephadex G-150. As cromatografias foram monitoradas através de leituras em espectrofotômetro no comprimento de onda de 280nm, dos líquidos coletados nos tubos. Os picos contendo a quimera foram coletados e submetidos à eletroforese (SDS-PAGE), sendo assim evidenciada a banda correspondente à mesma. Grupos de camundongos swiss foram imunizados com o vírus quimérico (CPSMV + p28), com o vírus CPSMV purificado e com a proteína p28 do CAEV, utilizando o adjuvante de Freund incompleto. Os anticorpos específicos produzidos contra o CPSMV e p28 reconheceram a proteína quimérica em Western Blotting e em teste de ELISA. Os anticorpos contra o vírus quimérico apresentaram títulos mais elevados do que os anticorpos produzidos contra a p28, demonstrando que o vírus quimérico apresenta maior imunogenicidade do que a proteína p28 sozinha. Os resultados mostraram que o acoplamento covalente entre o CPSMV e a p28 do CAEV foi obtido com sucesso, originando uma molécula estável não comprometendo a estrutura do capsídeo do CPSMV. Desta forma, sugere-se que o CPSMV possa ser utilizado como molécula carreadora na produção de vacinas para vírus que infectam animais.

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Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting)

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46651990000500013 ◽

1990 ◽

Vol 32 (5) ◽

pp. 379-383 ◽

Cited By ~ 3

Author(s):

Anna Maria Simonsen Stolf ◽

Eufrosina Setsu Umezawa ◽

Bianca Zingales

Keyword(s):

Trypanosoma Cruzi ◽

Western Blot ◽

Chagas Disease ◽

Western Blotting ◽

Specific Antibodies ◽

Internal Parasite ◽

Sds Page ◽

Blotting Technique ◽

Simultaneous Identification ◽

Acute Chagas Disease

A radioactive Western-blotting technique was developed by which the reactivity of Immunoglobulins (Igs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse T. cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgG specific antibodies. A different pattern of reactivity with acute Chagas' disease patients sera was thus obtained.

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High Expression of Human Amelogenin in E. Coli

Advances in Dental Research ◽

10.1177/08959374960100021201 ◽

1996 ◽

Vol 10 (2) ◽

pp. 187-194 ◽

Cited By ~ 6

Author(s):

D. Deutsch ◽

E. Chityat ◽

M. Hekmati ◽

A. Palmon ◽

Y. Farkash ◽

...

Keyword(s):

Amino Acid ◽

Western Blotting ◽

Rt Pcr ◽

Amino Acid Sequencing ◽

Terminal Amino Acid ◽

Expression Plasmid ◽

Over Expression ◽

E Coli ◽

Sds Page ◽

Terminal Amino

A human cDNA, encoding for the 175-aminoacid human amelogenin, was prepared by RT PCR from tooth bud mRNA and sub-cloned into pGEX-KG expression plasmid for over-expression in E. coli. The expressed protein was characterized by SDS-PAGE, Western blotting, and N-terminal amino acid sequencing.

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2D SDS PAGE in Combination with Western Blotting and Mass Spectrometry Is a Robust Method for Protein Analysis with Many Applications

Advances in Experimental Medicine and Biology - Advancements of Mass Spectrometry in Biomedical Research ◽

10.1007/978-3-030-15950-4_33 ◽

2019 ◽

pp. 563-574 ◽

Cited By ~ 2

Author(s):

Nancy Kendrick ◽

Costel C. Darie ◽

Matt Hoelter ◽

Ginny Powers ◽

Jon Johansen

Keyword(s):

Mass Spectrometry ◽

Western Blotting ◽

Protein Analysis ◽

Robust Method ◽

Sds Page

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Nachweis der Osteokalzinexpression osteoblastärer Zellen mandibulären Ursprungs, wachsend auf Biomaterialien, mittels RT-PCR und SDS-PAGE/Western Blotting

Mund- Kiefer- und Gesichtschirurgie ◽

10.1007/s10006-003-0495-7 ◽

2003 ◽

Vol 7 (5) ◽

pp. 294-300 ◽

Cited By ~ 6

Author(s):

D. Turhani ◽

C. Item ◽

D. Thurnher ◽

D. Kapral ◽

B. Cvikl ◽

...

Keyword(s):

Western Blotting ◽

Rt Pcr ◽

Sds Page

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The determination of immune reactive proteins inCysticercus tenuicolliscyst fluids by SDS-PAGE and Western blotting in sheep

Parasite ◽

10.1051/parasite/2003102141 ◽

2003 ◽

Vol 10 (2) ◽

pp. 141-145 ◽

Cited By ~ 1

Author(s):

M. Kara ◽

H.O. Sarimehmetoglu ◽

B. Gonenc

Keyword(s):

Western Blotting ◽

Sds Page

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ACTB and GAPDH proteins appear at multiple positions of SDS-PAGE and may not be suitable for serving as reference genes for the protein level determination in such techniques as Western blotting

10.1101/2020.03.05.978494 ◽

2020 ◽

Author(s):

Yan He ◽

Ju Zhang ◽

Jiayuan Qu ◽

Lucas Zellmer ◽

Yan Zhao ◽

...

Keyword(s):

Western Blotting ◽

Protein Level ◽

Reference Genes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Bioinformatic Analysis ◽

Protein Isoforms ◽

Leading Role ◽

Non Coding Rna ◽

Sds Page

AbstractMost human genes can produce multiple protein isoforms that should appear at multiple positions of polyacrylamide gel electrophoresis (PAGE) with sodium dodecyl sulfate (SDS), but most published results of Western blotting show only one protein. We performed SDS-PAGE of proteins from several human cell lines, isolated the proteins at the 72-, 55-, 48-, 40-, and 26-kD positions, and used liquid chromatography and tandem mass spectrometry (LC-MS/MS) to determine the protein identities. Although ACTB and GAPDH are 41.7-kD and 36-kD proteins, respectively, LC-MS/MS identified peptides of ACTB and GAPDH at all of these SDS-PAGE positions, making us wonder whether they produce some unknown protein isoforms. The NCBI (National Center for Biotechnology Information, USA) database lists only one ACTB mRNA but five GAPDH mRNAs and one non-coding RNA. The five GAPDH mRNAs encode three protein isoforms, while our bioinformatic analysis identified a 17.6-kD isoform encoded by the non-coding RNA. All LC-MS/MS-identified GAPDH peptides at all positions studied are unique, but some of the identified ACTB peptides are shared by ACTC1, ACTBL2, POTEF, POTEE, POTEI, and POTEJ. ACTC1 and ACTBL2 belong to the ACT family with great similarities to ACTB in protein sequence, whereas the four POTEs are ACTB-containing chimeric genes with the C-terminus of their proteins highly similar to ACTB. These data collectively disqualify GAPDH and ACTB from serving as the reference genes for determination of the protein level in such techniques as Western blotting, a leading role these two genes have been playing for decades in the biomedical research.

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