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

ISRN Urology ◽  
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.

Tetrodotoxin-resistant release of ATP from superfused rabbit detrusor muscle during electrical field stimulation in the presence of luciferin–luciferase

1984 ◽  
Vol 62 (1) ◽  
pp. 153-156 ◽  
Author(s):  
Archana Chaudhry ◽  
John W. Downie ◽  
Thomas D. White
Keyword(s):  
Action Potentials ◽  
Electrical Field Stimulation ◽  
Detrusor Muscle ◽  
Electrical Field ◽  
Stimulation Parameters ◽  
Rabbit Bladder ◽  
Firefly Luciferin ◽  
Rabbit Urinary Bladder ◽  
Stimulation Of

The present study was carried out to assess the possible role of ATP in the noncholinergic, nonadrenergic transmission in the rabbit urinary bladder. When rabbit detrusor muscle strips were superfused with medium containing firefly luciferin–luciferase and stimulated transmurally at low stimulation parameters, tetrodotoxin-sensitive contractions were obtained but no release of ATP could be detected. However, at somewhat higher stimulation parameters, release of ATP was observed. This release of ATP was not diminished by tetrodotoxin indicating that ATP was not likely released as a result of propagated action potentials in nerves. Because contractions persisted in the presence of tetrodotoxin, it is possible that the ATP might have been released as a result of direct electrical stimulation of the muscle. These results do not support the idea that ATP is released as a neurotransmitter in the rabbit bladder.

scholarly journals Generating and characterizing the anti-human CD45 monoclonal antibody

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.

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

2005 ◽  
Vol 35 (6) ◽  
pp. 1363-1367 ◽  
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.

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

1990 ◽  
Vol 32 (5) ◽  
pp. 379-383 ◽  
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.

High Expression of Human Amelogenin in E. Coli

1996 ◽  
Vol 10 (2) ◽  
pp. 187-194 ◽  
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.

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

2003 ◽  
Vol 7 (5) ◽  
pp. 294-300 ◽  
Author(s):  
D. Turhani ◽  
C. Item ◽  
D. Thurnher ◽  
D. Kapral ◽  
B. Cvikl ◽  
...  
Keyword(s):  
Western Blotting ◽  
Rt Pcr ◽  
Sds Page
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