A imunoprecipitação como método de purificação de nitrotirosinas
Data
2019
Autores
Pelitz, Bianca Rodrigues Cardoso [UNIFESP]
Orientadores
Fernandes, Liliam [UNIFESP]
Tipo
Trabalho de conclusão de curso de graduação
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A imunoprecipitação, considerada uma adaptação da cromatografia de afinidade,
utiliza anticorpos de alta especificidade para isolar proteínas alvo (antígenos). A combinação dessa técnica com a análise por Western Blot fornece maior especificidade para identificar proteínas de baixa ocorrência, além de aumentar a sensibilidade da detecção. As nitrotirosinas são proteínas significantes no processo de estresse oxidativo e nitrosativo. Nos vasos sanguíneos, a nitração de resíduos de tirosina a partir de espécies reativas de oxigênio e de nitrogênio pode levar a alterações importantes na sinalização celular, promovendo a disfunção endotelial e consequente prejuízo a toda a rede circulatória. Nesse contexto, o presente trabalho descreve detalhadamente a sinalização redox e a geração de nitrotirosinas no endotélio vascular, e, paralelamente, apresenta e comenta a implantação da técnica de imunoprecipitação de nitrotirosinas em células endoteliais em cultura, seguida da detecção dessas proteínas por método de Western Blot. Inicialmente as culturas foram subcultivadas e, ao atingirem estágio de semiconfluência, foram expostas a agentes vasoativos durante 24 horas. Os agentes empregados foram: 1) Angiotensina II, um peptídeo endógeno vasoconstritor, descrito na literatura como um agente pró-oxidante em vários tipos celulares, e 2) Luteolina, um flavonoide da classe das flavonas, presente em vários alimentos, e descrito na literatura como um agente antioxidante. As culturas celulares foram então coletadas, as proteínas totais foram quantificadas, e as amostras foram submetidas à imunoprecipitação em gel de agarose previamente incorporado com anticorpos anti-nitrotirosina. A quantidade de proteínas nas amostras se mostrou adequada para o prosseguimento do ensaio, demonstrando que medição foi realizada de maneira correta. A fração eluída foi então submetida a eletroforese, seguida de transferência para membrana de nitrocelulose e posterior incubação com os anticorpos primário (anti-nitrotirosina) e secundário correspondente. A separação das proteínas ocorreu de forma satisfatória e foi possível observar o marcador de peso molecular na membrana de nitrocelulose, indicando que o procedimento de transferência do Western Blot foi realizado com sucesso. A revelação da membrana foi feita por quimioluminescência e detectada em fotodocumentador. A revelação demonstrou que o método de imunoprecipitação de nitrotirosinas foi implantado com sucesso: a marcação de peso molecular foi bem-sucedida, assim como os controles negativo e positivo. Foram detectadas marcações em todas as amostras experimentais. Todos os passos do procedimento foram bem definidos e adequadamente estabelecidos. A metodologia da imunoprecipitação de nitrotirosinas em células endoteliais cultivadas foi implantada e padronizada com sucesso. Essa abordagem será valiosa no estudo da ação de substâncias pró e antioxidantes sobre o endotélio vascular, e poderá ser empregada em futuros projetos do Laboratório de Farmacologia Vascular.
Immunoprecipitation, considered an adaptation of affinity chromatography, uses antibodies of high specificity to isolate target proteins (antigens). The combination of this technique with Western Blot analysis provides greater specificity to identify low occurrence proteins as well as increases detection sensitivity. Nitrotyrosines are significant proteins in the process of oxidative and nitrosative stress. In the blood vessels nitration of tyrosine residues from oxygen and nitrogen reactive species can lead to important alterations in cellular signaling, promoting endothelial dysfunction and consequent damage to the entire circulatory network. In this context, the present work describes in detail the redox signaling and the generation of nitrotyrosines in the vascular endothelium, and, in parallel, presents and comments the implantation of the immunoprecipitation technique of nitrotyrosines in endothelial cells in culture, followed by detection of these proteins by Western Blot method. Initially the cultures were sub cultured and, upon reaching a semi confluence stage, were exposed to vasoactive agents for 24 hours. The agents used were: 1) Angiotensin II, an endogenous vasoconstrictor peptide, described in literature as a pro-oxidant agent in several cell types, and 2) Luteolin, a flavonoid of the flavone class present in several foods, and described in literature as an antioxidant agent. Cell cultures were then collected, total proteins quantified, and samples were subjected to immunoprecipitation with agarose gel previously incorporated with anti-nitrotyrosine antibodies. The amount of protein in the samples proved to be adequate for the continuation of the assay, demonstrating that measurement was performed correctly. The eluted fraction was then subjected to electrophoresis, followed by transfer to nitrocellulose membrane and subsequent incubation with the corresponding primary antibody (anti-nitrotyrosine) and corresponding secondary antibody. Protein separation occurred satisfactorily and it was possible to observe the molecular weight marker on the nitrocellulose membrane, indicating that the Western Blot transfer procedure was successfully performed. The membrane was revealed by chemiluminescence and detected by photodocumentation. The revelation demonstrated that the nitrotyrosine immunoprecipitation method was successfully implanted: molecular weight marking was successful, as the negative and positive controls. Markers were detected in all experimental samples. All steps of the procedure were well defined and adequately established. The methodology of immunoprecipitation of nitrotyrosines in cultured endothelial cells was successfully implanted and standardized. This approach will be valuable in the study of the action of pro-and antioxidant substances on the vascular endothelium, and may be used in future projects of the Laboratory of Vascular Pharmacology.
Immunoprecipitation, considered an adaptation of affinity chromatography, uses antibodies of high specificity to isolate target proteins (antigens). The combination of this technique with Western Blot analysis provides greater specificity to identify low occurrence proteins as well as increases detection sensitivity. Nitrotyrosines are significant proteins in the process of oxidative and nitrosative stress. In the blood vessels nitration of tyrosine residues from oxygen and nitrogen reactive species can lead to important alterations in cellular signaling, promoting endothelial dysfunction and consequent damage to the entire circulatory network. In this context, the present work describes in detail the redox signaling and the generation of nitrotyrosines in the vascular endothelium, and, in parallel, presents and comments the implantation of the immunoprecipitation technique of nitrotyrosines in endothelial cells in culture, followed by detection of these proteins by Western Blot method. Initially the cultures were sub cultured and, upon reaching a semi confluence stage, were exposed to vasoactive agents for 24 hours. The agents used were: 1) Angiotensin II, an endogenous vasoconstrictor peptide, described in literature as a pro-oxidant agent in several cell types, and 2) Luteolin, a flavonoid of the flavone class present in several foods, and described in literature as an antioxidant agent. Cell cultures were then collected, total proteins quantified, and samples were subjected to immunoprecipitation with agarose gel previously incorporated with anti-nitrotyrosine antibodies. The amount of protein in the samples proved to be adequate for the continuation of the assay, demonstrating that measurement was performed correctly. The eluted fraction was then subjected to electrophoresis, followed by transfer to nitrocellulose membrane and subsequent incubation with the corresponding primary antibody (anti-nitrotyrosine) and corresponding secondary antibody. Protein separation occurred satisfactorily and it was possible to observe the molecular weight marker on the nitrocellulose membrane, indicating that the Western Blot transfer procedure was successfully performed. The membrane was revealed by chemiluminescence and detected by photodocumentation. The revelation demonstrated that the nitrotyrosine immunoprecipitation method was successfully implanted: molecular weight marking was successful, as the negative and positive controls. Markers were detected in all experimental samples. All steps of the procedure were well defined and adequately established. The methodology of immunoprecipitation of nitrotyrosines in cultured endothelial cells was successfully implanted and standardized. This approach will be valuable in the study of the action of pro-and antioxidant substances on the vascular endothelium, and may be used in future projects of the Laboratory of Vascular Pharmacology.