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

Parasite ◽  
2003 ◽  
Vol 10 (2) ◽  
pp. 141-145 ◽  
Author(s):  
M. Kara ◽  
H.O. Sarimehmetoglu ◽  
B. Gonenc
Keyword(s):  
Western Blotting ◽  
Sds Page

Characterization of Glycoprotein Fraction from Carp Pituitaries Isolated Using Concanavalin A as the Affinity Ligand

1998 ◽  
Vol 63 (3) ◽  
pp. 434-440 ◽  
Author(s):  
Irena Hulová ◽  
Jana Barthová ◽  
Helena Ryšlavá ◽  
Václav Kašička
Keyword(s):  
Concanavalin A ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Gel Chromatography ◽  
Affinity Ligand ◽  
Sds Page ◽  
Terminal Amino ◽  
Α And Β Subunits

Glycoproteins that have affinity to Concanavalin A were isolated from the acetone-dried pituitaries of common carp (Cyprinus carpio L.). Two fractions of glycoproteins were separated using gel chromatography on Superdex 75HR. The fraction with lower molecular weight (30 000) corresponding to the carp gonadotropin cGtH II was composed of two subunits as determined using SDS-PAGE. This protein fraction was further divided into four components using reversed-phase HPLC. Two fractions were pure α and β subunits of cGtH II as follows from immunodetection and from determination of N-terminal amino acid sequences. The other two were a mixture of α and β subunits as was also revealed by N-terminal analysis. Capillary electrophoresis was also used for characterization of isolated glycoproteins.

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.

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 Optimization of Chemoenzymatic Mass Tagging by Strain-Promoted Cycloaddition (SPAAC) for the Determination of O-GlcNAc Stoichiometry by Western Blotting

Biochemistry ◽  
2018 ◽  
Vol 57 (40) ◽  
pp. 5769-5774 ◽  
Author(s):  
Narek Darabedian ◽  
John W. Thompson ◽  
Kelly N. Chuh ◽  
Linda C. Hsieh-Wilson ◽  
Matthew R. Pratt
Keyword(s):  
Western Blotting

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.

scholarly journals Effect of anti-thyroid peroxidase (TPO) antibodies on TPO activity measured by chemiluminescence assay

1997 ◽  
Vol 43 (8) ◽  
pp. 1392-1396 ◽  
Author(s):  
V Kaczur ◽  
Gy Vereb ◽  
I Molnár ◽  
G Krajczár ◽  
E Kiss ◽  
...  
Keyword(s):  
Western Blotting ◽  
Competitive Inhibition ◽  
High Sensitivity ◽  
Inhibitory Effect ◽  
Thyroid Peroxidase ◽  
Chemiluminescence Detection ◽  
Chemiluminescence Method ◽  
Blotting Technique ◽  
Hypothyroid Patients

Abstract A chemiluminescence method was developed to measure thyroid peroxidase (TPO) activity and the inhibitory effect of anti-TPO antibodies in purified porcine TPO. The TPO preparation was characterized kinetically and controlled by Western-blotting technique. The chemiluminescence method proved to be reproducible and much more sensitive than the widely used guaiacol method, being able to detect TPO concentrations of 2.21 × 10−5 g/L vs 6.63 × 10−2g/L with the latter. Otherwise, the determinations with the two methods correlated well (r = 0.76). Investigating the effect of IgGs from 23 hypothyroid patients on measured TPO activity, we detected inhibition in 19 cases with the chemiluminescence technique (15 with the guaiacol method). Anti-TPO antibodies showed competitive inhibition of TPO activity with respect to the substrate guaiacol. In both systems, the inhibition is present in the IgG F(ab′)2 fragment. We conclude that the high sensitivity of chemiluminescence detection allows routine determination of the inhibition of TPO activity by anti-TPO antibodies.

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.

scholarly journals Sarcoplasmatic and myofibrillar protein changes caused by acute heat stress in broiler chicken

2008 ◽  
Vol 65 (5) ◽  
pp. 453-458 ◽  
Author(s):  
Carolina de Castro Santos ◽  
Eduardo Francisquine Delgado ◽  
José Fernando Machado Menten ◽  
Aparecida Carla de Moura Pedreira ◽  
Carmen Josefina Contreras Castillo ◽  
...  
Keyword(s):  
Heat Stress ◽  
Broiler Chicken ◽  
Myofibrillar Protein ◽  
Environmental Condition ◽  
Fragmentation Index ◽  
Acute Heat Stress ◽  
Sds Page ◽  
And Migration ◽  
Water Holding

Acute heat stress (AHS) modifies the structure of myofibrils affecting functional properties of meat, mainly the water holding capacity. This experiment aimed to identify changes in proteolysis and migration between the myofibrillar and sarcoplasmatic fractions due to pre-slaughter AHS. Myofibrillar fragmentation index (MFI), SDS-PAGE, western blot of vinculin (WB) and shear force (SF) were determined. Six hundred broilers (Gallus gallus) were slaughtered in three different days (ST). In each ST, groups of ten animals were placed in transport crates and submitted to AHS (35ºC, 75 - 85% RH) for 2 hours. Simultaneously, the non-stressed broilers (NS) were kept in thermoneutral environment (22ºC, 83 ± 6.6% RH) within the crates in the same density. After slaughter, the breast muscles were kept refrigerated until the withdrawal of all samples (0, 1, 2, 6 and 24 hours after slaughter). Sampling within AHS and NS birds was collected according to lightness value (normal L* < 49, and high > 51), except for determination of MFI and SF. The lightness was used later to perform SDS-PAGE and WB analyses. MFI kinetics showed that the fragmentation rate was superior in animals NS, indicating that AHS can harm proteolysis and rate of myofibrillar fragmentation. However, the extent of fragmentation did not change, as well as SF values. SDS-PAGE for Troponin fragments indicated a differentiated pattern between AHS and NS. The WB did not show alterations in vinculin fragmentation. Modifications in sarcoplasmatic fraction are observed in meat with high L*values, independent of environmental condition.

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