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Master's Dissertation
DOI
https://doi.org/10.11606/D.10.2007.tde-21052007-125151
Document
Author
Full name
Fabiana Santos Matsumoto
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2007
Supervisor
Committee
Miglino, Maria Angélica (President)
Ambrosio, Carlos Eduardo
Silva, Tatiana Jazedje da Costa
Title in Portuguese
Caracterização das proteínas do saco vitelínico de embriões bovinos Bos indicus
Keywords in Portuguese
Western Blot
Alfa-1 antitripsina
Alfafetoproteina
Proteínas
Saco Vitelínico
Transferrina
Abstract in Portuguese
O saco vitelínico é uma das membranas embrionárias que desempenham um papel importante para a sobrevivência inicial do embrião em muitas espécies de mamíferos, além de produzir proteínas necessárias para o desenvolvimento do mesmo. Foram coletados 17 embriões bovinos, em diferentes períodos gestacionais afim de identificar as proteínas alfafetoproteína, alfa- 1 antitripsina e transferrina, presentes no saco vitelínico destes,para tanto realizou-se a técnica de Western Blot com eletroforese em gel de poliacrilamida, SDS-PAGE a 6%. Os géis, após a corrida, foram corados com Comassie blue, e as membranas de nitrocelulose, após a transferência, com Ponceau. Utilizaram-se os anticorpos monoclonal para alfafetoproteína anti-camundongo, monoclonal, receptpr de transferrin anti-camundongo IgG1, e policlonal para alfa- 1 antitripsina anti-coelho como anticorpos primário e conjugado para peroxidase e fosfatase como secundários. A revelação foi do tipo colorimétrica-fosfatase alcalina e por ECL. O saco vitelínico apresentou-se bem desenvolvido até os 50 dias de gestação, onde, a partir desse período o processo de involução está bem caracterizado Em algumas amostras do saco vitelínico detectamos a presença da alfafetoproteina, alfa-1 antitripsina e da transferrina, porém em algumas amostras as bandas estavam fracas, mostrando assim, que os anticorpos reagem com as proteínas bovinas. O fato de aparecerem bandas fracas pode estar relacionado a uma fraca reação cruzada por se tratar de um anticorpo não específico.
Title in English
Characterization of the yolk sac proteins of the Bos indicus bovine embryos
Keywords in English
Western Blot
Alfa 1- antitrypsin
Alfafetoprotein
Proteins
Transferrin
Yolk sac
Abstract in English
In many species of mammals, the yolk sac is one of the embrionary membranes that plays an important role in the embryo´s initial survival, as well as, in the manufacturing of the necessary proteins for its development. In order to identify the proteins: alfafetoprotein, alfa 1 - antitrypsin, and transferrin present in the cow´s embryo´s yolk sac, 17 bovine embryos were collected in different pregnancy periods. This procedure was performed by Western Blot Technique with a polyacrylamide gel electrophoresis, SDS-PAGE, at 6%. Gels following the electrophoresis, where tainted with Comassie blue, and the membranes of Nitrocellulose, following their transference (the proteins that were present in the gel go to the membrane), with Ponceau. Monoclonal Antibody mouse anti human α-fetoprotein, alphafetoprotein mouse monoclonal antibody, transferrin receptor mouse IgG1, and rabbit polyclonal to alpha 1 antitrypsin were used as primary antibodies, and Peroxidase labelled antimouse e Peroxidase labelled antirabbit e anti-mouse IgG- Alkaline Fosfatase as secundary ones. The membrane´s revelation was of the alcaline fosfatase colormetric type and by ECL. The yolk sac was presented well developed until the 50 days of gestation, where to break of this period the involution process well it is characterized. In some of the yolk sac samples we detected the presence of alfafetoprotein, alfa 1- antitrypsin, and transferrin, however, the bands in some specimens (samples) were weak, demonstrating that the antibodies react with the bovine proteins. The fact that weak bands appeared might be related to a weak cross reaction since we are dealing with a non specific antibody.
 
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Publishing Date
2007-05-29
 
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