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

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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CLONAGEM, EXPRESSÃO E CARACTERIZAÇÃO DA NUCLEOPROTEÍNA RECOMBINANTE DO VÍRUS DA BRONQUITE INFECCIOSA EM ESCHERICHIA COLI E EM PICHIA PASTORIS

Arquivos do Instituto Biológico ◽

10.1590/1808-1657v77p0012010 ◽

2010 ◽

Vol 77 (1) ◽

pp. 1-9

Author(s):

A.M. Gibertoni ◽

M.C.M. Gonçalves ◽

M.F.S. Montassier ◽

C.C. Fernandes ◽

H.J. Montassier

Keyword(s):

Escherichia Coli ◽

Pichia Pastoris ◽

Western Blotting ◽

Rt Pcr ◽

E Coli ◽

Sds Page

RESUMO O gene da proteína de nucleocapsídeo (1.230 pb) da estirpe M41 do vírus da bronquite infecciosa (VBI) foi amplificado pelas reações de transcrição reversa e em cadeia da polimerase (RT-PCR) e clonado, em seguida, em dois sistemas; pET28a - Escherichia coli e pFLD -Pichia pastoris. Os produtos recombinantes construídos para expressão (pET28a-N ou pFLD-N) foram identificados por análises de PCR e de sequenciamento de nucleotídeos. Os clones transformantes da linhagem BL21 de E. coli e da linhagem GS115 de P. pastoris foram submetidos aos protocolos apropriados de indução. A expressão da proteína N de fusão com etiqueta de poli-histidina e com massa molecular de 54 kDa foi determinada pelas técnicas de SDS-PAGE e de Western blotting, confirmando-se que ambas proteínas N recombinantes apresentaram tamanhos e antigenicidade compatíveis com a proteína N nativa do próprio VBI. O sistema E. coli expressou uma quantidade relevante da proteína N recombinante, enquanto que o sistema P. pastoris produziu uma baixa recuperação dessa proteína recombinante. A proteína N recombinante gerada pelo sistema bacteriano foi purificada em resina de níquel-sepharose. O conjunto de resultados indica que o sistema de expressão constituído por pET28a – E. coli é mais efetivo para produzir a proteína N recombinante do VBI destinada ao uso como antígeno para detectar anticorpos anti-virais específicos em ensaios de imunodiagnóstico para essa infecção viral.

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323 EXPRESSION OF PSP-I AND PSP-II IN THE REPRODUCTIVE TRACT OF THE BOAR BY IMMUNOHISTOCHEMISTRY, WESTERN BLOTTING, AND RT-PCR

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab323 ◽

2007 ◽

Vol 19 (1) ◽

pp. 277

Author(s):

E. M. Garcia ◽

J. M. Vazquez ◽

I. Parrilla ◽

J. J. Calvete ◽

L. Sanz ◽

...

Keyword(s):

Western Blotting ◽

Reproductive Tract ◽

Polyclonal Antibodies ◽

Seminal Vesicles ◽

Lower Amount ◽

Rt Pcr ◽

Variable Intensity ◽

Glass Slides ◽

Sds Page ◽

Boar Spermatozoa

In boar seminal plasma, PSP-I and PSP-II form a noncovalent heterodimer, which is known to have a beneficial effect on highly extended boar spermatozoa. These proteins are mainly produced by the seminal vesicles of the male reproductive tract and are mixed with the spermatozoa during ejaculation. This study assessed the epithelial localization and expression of spermadhesin PSP-I and PSP-II subunits using immunohistochemical, western blotting, and RT-PCR methods in porcine testis, epididymis (caput, corpus, and caudal), seminal vesicles, and bulbourethral glands. Tissues were collected from 10 mature boars (Swedish Yorkshire) of proven fertility, frozen or fixed, and embedded in paraffin. Sections of 5 �m were mounted on poly-l-lysine-coated glass slides for immunohistochemistry, and 50 mg from the frozen counterparts were homogenized to isolate proteins (Western blotting study) or RNA (RT-PCR study). Polyclonal antibodies (antiPSP-I and antiPSP-II) against proteins (PSP-I and PSP-II) were used. The immunohistochemistry showed positive labelling for both antibodies in the epithelium of seminal vesicles in all males. Positive immunolabelling, but of variable intensity, was present in the epididymal epithelium (caput, corpus, and caudal), with variation among segments and boars. No labelling was found in the seminiferous epithelium or bulbourethral glands of any boar. After SDS-PAGE and Western blotting, immunoreactive bands were obtained in the extracts of all tissues for both PSP proteins. Among tissues, the highest intensity corresponded to the seminal vesicle lane for both proteins. Epididymis (caput, corpus, and caudal), bulbourethral gland, and testis showed more feeble bands, yet present. The intensity of the bands varied among boars. Amplification products from mRNA were obtained in all tissues explored by RT-PCR, using specific primers for PSP-I and PSP-II. The same trend was obtained regarding intensity bands, corresponding with the intensity obtained by Western blotting. The results indicate that PSP-I and PSP-II are mainly expressed in seminal vesicles and epididymal segments and, in lower amount, in the testis and bulbourethral gland. This work was supported by Formas, Stockholm, and Fundaci�n S�neca, Murcia, Spain.

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Nitrotyrosine Density of Rabbit Urinary Bladder Muscle and Mucosa Measured via Western Blotting and 96-Well Plate Analysis

ISRN Urology ◽

10.5402/2012/618247 ◽

2012 ◽

Vol 2012 ◽

pp. 1-5

Author(s):

Brittany Fitzpatrick ◽

Catherine Schuler ◽

Robert E. Leggett ◽

Robert M. Levin

Keyword(s):

Quantitative Analysis ◽

Western Blotting ◽

Optical Density ◽

Tissue Concentration ◽

Detrusor Muscle ◽

Pvdf Membrane ◽

Sds Page ◽

Wash Buffer ◽

Rabbit Bladder ◽

Plate Analysis

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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The Progesterone Receptor in Human Term Amniochorion and Placenta Is Isoform C

Endocrinology ◽

10.1210/en.2005-0510 ◽

2006 ◽

Vol 147 (2) ◽

pp. 687-693 ◽

Cited By ~ 20

Author(s):

Anthony H. Taylor ◽

Penny C. McParland ◽

David J. Taylor ◽

Stephen C. Bell

Keyword(s):

Progesterone Receptor ◽

Western Blotting ◽

Cell Types ◽

Specific Role ◽

Fetal Membranes ◽

Rt Pcr ◽

Reproductive Tissues ◽

Pcr Techniques ◽

Human Parturition ◽

Extraembryonic Tissues

The mechanism that initiates human parturition has been proposed to be functional progesterone withdrawal whereby the 116-kDa B isoform of the progesterone receptor (PR-B) switches in favor of the 94-kDa A isoform (PR-A) in reproductive tissues. Recently other PR isoforms, PR-S, PR-C, and PR-M generated from the same gene have been identified and partially characterized. Using immunohistochemical, Western blotting, and RT-PCR techniques, evidence is provided that the major PR isoform present in human term fetal membranes (amnion and chorion) and syncytiotrophoblast of the placenta is neither of the classical nuclear PR-B or PR-A isoforms but is the N terminally truncated 60-kDa PR-C isoform. Evidence is also provided that the PR-C isoform resides in the cytoplasm of the expressing cell types. Data are also presented to show that PR-B, PR-A, and PR-S isoforms are essentially absent from the amnion and chorion, whereas PR isoforms A, B, C, and S are all present in the decidua, with PR-A being the major isoform. The syncytiotrophoblast of the placenta contains the cytoplasmic PR-C isoform but not PR-A, PR-B, or PR-S. The major PR isoform in the amnion, chorion, and placenta is PR-C, suggesting that the cytoplasmic PR-C isoform has a specific role in extraembryonic tissues and may be involved in the regulation of human parturition.

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Expression of β-catenin in human trophoblast and its role in placenta accreta and placenta previa

Journal of International Medical Research ◽

10.1177/0300060518799265 ◽

2018 ◽

Vol 47 (1) ◽

pp. 206-214

Author(s):

Qing Han ◽

Lianghui Zheng ◽

Zhaodong Liu ◽

Jinying Luo ◽

Rongxin Chen ◽

...

Keyword(s):

Western Blotting ◽

Cell Invasion ◽

Placenta Accreta ◽

Placenta Previa ◽

Control Group ◽

Rt Pcr ◽

Lesion Area ◽

Human Trophoblast ◽

Chorionic Villi ◽

Pcr Techniques

Objectives To investigate the expression of β-catenin in chorionic villi, and to explore its roles in placenta accreta and placenta previa. Methods We compared β-catenin expression in the control group, placenta accreta group (lesion area and normal zones), and placenta previa group (placental central and placental edge zones) by immunohistochemistry, Western blotting, and RT-PCR techniques. Results Compared with the normal group, the placenta accreta group had a longer length of stay, greater bleeding volume, and lower newborn birth weight. Further, the expression of β-catenin was lower in both placenta previa and placenta accreta groups than in the control group, as measured by immunohistochemistry. Compared with the control group, expression of β-catenin was significantly lower in the placenta previa and placenta accreta groups by Western blotting and RT-PCR. Importantly, the level of placental β-catenin was significantly different when compared between the lesion and normal zones of placenta. Conclusion The expression of β-catenin in placenta accreta might play an important role in the regulation of placental cell invasion; low expression of β-catenin in placenta accreta might be responsible for excessive trophoblastic invasion.

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Μελέτη γλυκοζαμινογλυκανών και πρωτεογλυκανών σε λευχαιμικά κύτταρα

10.12681/eadd/11230 ◽

1998 ◽

Author(s):

Ευδοκία Μακατσώρη

Keyword(s):

Western Blotting ◽

Rt Pcr

Η ΠΑΡΟΥΣΑ ΔΙΑΤΡΙΒΗ ΕΙΧΕ ΣΤΟΧΟ ΤΗ ΜΕΛΕΤΗ ΠΡΩΤΕΟΓΛΥΚΑΝΩΝ (PGS)ΚΑΙ ΓΛΥΚΟΖΑΜΙΝΟΓΛΥΚΑΝΩΝ (GAGS)ΛΕΥΧΑΙΜΙΚΩΝ ΛΕΜΦΟΚΥΤΤΑΡΩΝ ΚΑΙ ΜΟΝΟΚΥΤΤΑΡΩΝ ΚΑΙ ΤΗ ΜΕΛΕΤΗ ΤΗΣ ΔΡΑΣΗΣ ΜΙΤΟΓΟΝΩΝ ΣΤΗ ΣΥΝΘΕΣΗ ΚΑΙ ΚΑΤΑΝΟΜΗ ΤΩΝ GAGS ΟΠΩΣ ΕΠΙΣΗΣ ΤΗ ΜΕΛΕΤΗ ΔΡΑΣΗΣ ΕΞΩΓΕΝΩΝ GAGS ΣΤΟΝ ΚΥΤΤΑΡΙΚΟ ΠΟΛΛΑΠΛΑΣΙΑΣΜΟ,ΜΟΝΩΝ ΤΟΥΣ,Η ΣΕ ΣΥΝΔΥΑΣΜΟ ΜΕ ΜΙΤΟΓΟΝΑ.ΕΠΙΛΕΧΘΗΚΑΝ ΤΡΕΙΣ ΛΕΥΧΑΙΜΙΚΕΣ ΚΥΤΤΑΡΙΚΕΣ ΣΕΙΡΕΣ :JURKAT (ΟΞΕΙΑ Τ-ΛΕΜΦΟΚΥΤΤΑΡΙΚΗ ΛΕΥΧΑΙΜΙΑ),DAUDI (Β-ΛΕΜΦΟΚΥΤΤΑΡΙΚΗ ΛΕΥΧΑΙΜΙΑ)ΚΑΙ THP-1 (ΟΞΕΙΑ ΜΟΝΟΚΥΤΤΑΡΙΚΗ ΛΕΥΧΑΙΜΙΑ).ΑΡΧΙΚΑ ΜΕ RT-PCR ΜΕΛΕΤΗΘΗΚΕ Η ΕΜΦΑΣΗ ΠΡΩΤΕΟΓΛΥΚΑΝΩΝ ΣΕ ΕΠΙΠΕΔΟ MRNA,ΚΑΙ ΒΡΕΘΗΚΕ ΟΤΙ ΚΑΙ ΟΙ ΤΡΕΙΣ ΚΥΤΑΡΙΚΕΣ ΣΕΙΡΕΣ ΕΧΟΥΝ ΤΗ ΔΥΝΑΤΟΤΗΤΑ ΣΥΝΘΕΣΗΣ ΣΥΝΔΕΙΝΗΣ-1,-2,-4,ΓΛΥΠΙΚΑΝΗΣ,CD44,ΒΕΡΣΙΚΑΝΗΣ-0 ΚΑΙ ΒΕΡΣΙΚΑΝΗΣ-1(ΤΥΠΟΣΕΝΑΛΛΑΚΤΙΚΟΥ ΜΑΤΙΣΜΑΤΟΣ ΤΗΣ ΒΕΡΣΙΚΑΝΗΣ). ΤΑ DAUDI ΕΧΟΥΝ ΕΠΙΠΛΕΟΝ ΤΗ ΔΥΝΑΤΟΤΗΤΑ ΣΥΝΘΕΣΗΣ ΙΝΟΜΟΝΤΟΥΛΙΝΗΣ,ΕΝΩ ΤΑ THP-1 ΜΠΟΡΕΙ ΝΑ ΣΥΝΘΕΤΟΥΝ ΠΕΡΛΕΚΑΝΗ ΚΑΙ ΘΡΟΜΒΟΜΟΝΤΟΥΛΙΝΗ. ΠΙΣΤΟΠΟΙΗΘΗΚΕ ΜΕ WESTERN BLOTTING Η ΠΑΡΟΥΣΙΑ ΣΥΝΔΕΙΝΗΣ -1,ΔΥΟΤΥΠΩΝ ΣΥΝΔΕΙΝΗΣ -4 ΔΙΑΦΟΡΕΤΙΚΗΣ ΓΛΥΚΟΖΙΛΙΩΣΗΣ,Η ΜΕΓΑΛΥΤΕΡΗΣ ΘΕΙΩΣΗΣ,ΘΡΟΜΒΟΜΟΝΤΟΥΛΙΝΗΣ, ΒΕΡΣΙΚΑΝΗΣ-0,ΒΕΡΣΙΚΑΝΗΣ-1 ΜΙΑΣ PG ΘΕΙΙΚΗΣ ΚΕΡΑΤΑΝΗΣ ΚΑΙ ΠΕΡΛΕΚΑΝΗΣΑΝΑΛΟΓΑ ΜΕ ΤΗΝ ΚΑΘΕ ΚΥΤΤΑΡΙΚΗ ΣΕΙΡΑ.ΜΕ ΧΡΗΣΗ ΕΝΖΥΜΙΚΩΝ ΚΑΤΕΡΓΑΣΙΩΝ ΚΑΙ ΑΝΑΛΥΣΗΣ HPLC ΒΡΕΘΗΚΕ ΠΩΣ ΚΑΜΙΑ ΚΥΤΤΑΡΙΚΗ ΣΕΙΡΑ ΔΕ ΣΥΝΘΕΤΕΙ HA.ΣΥΝΘΕΤΟΥΝ ΚΥΡΙΩΣ CSΚΑΙ ΣΕ ΜΙΚΡΟΤΕΡΟ ΠΟΣΟΣΤΟ HS.ΑΚΟΛΟΥΘΩΣ,ΕΛΕΓΘΗΚΕ Η ΕΠΙΔΡΑΣΗ ΕΞΩΓΕΝΩΝ GAGS ΣΤΟΝ ΠΟΛΛΑΠΛΑΣΙΑΣΜΟ ΤΩΝ ΚΥΤΤΑΡΩΝ,ΟΠΩΣ ΕΠΙΣΗΣ ΚΑΙ Η ΔΡΑΣΗ ΓΛΥΚΟΖΑΜΙΝΟΓΛΥΚΑΝΩΝ ΠΑΡΟΥΣΙΑ ΜΙΤΟΓΟΤΩΝ ΣΤΟΝ ΠΟΛΛΑΠΛΑΣΙΑΣΜΟ ΤΩΝ ΚΥΤΤΑΡΩΝ ,ΜΕ ΣΚΟΠΟ ΝΑ ΔΙΑΠΙΣΤΩΘΕΙ ΤΟ ΚΑΤΑ ΠΟΣΟ ΣΥΜΜΕΤΕΧΟΥΝ ΟΙ GAGS ΣΤΟΥΣ ΜΗΧΑΝΙΣΜΟΥΣ ΔΙΕΓΕΡΣΗΣ.

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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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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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