Abstract
Analysis of the posttranslational modification of proteins, such as phosphorylation, might yield misleading results due to the presence of other proteins with similar electrophoretic properties that coimmunoprecipitate with the target protein. The aim of the present work was to develop a reliable, easy and economical technique to completely isolate a protein from its complex. Here we present a new assay developed to fully isolate proteins from macromolecular complexes that consists of an initial SDS/PAGE (under reducing conditions), which isolates the target protein, followed by transfer of the proteins to a buffer, from which the target protein is recaptured by conventional immunoprecipitation. This technique, that we have termed “Protein Complex Immunological Separation Assay” (ProCISA), successfully separated proteins of different sizes, such as pp60Src and the IP3 receptor (IP3R), from their complexes. We show that ProCISA allows the investigation of the tyrosine phosphorylation state of isolated proteins. This technique could also be used to study other posttranslational modifications without risk of misleading results resulting from contamination with other proteins of similar electrophoretic mobility which complex with the protein of interest.
Resumen
El análisis de las modificaciones postraduccionales de las proteínas, como la fosforilación, puede proporcionar resultados erróneos por la presencia de proteínas con semejanzas electroforéticas que coinmunoprecipitan con la proteína diana. El objetivo del presente trabajo consistió en desarrollar una técnica económica, sencilla y fiable que permitiese realizar la separación de las proteínas pertenecientes a un complejo proteico. Hemos desarrollado una técnica denominada ProCISA (“Protein Complex Immunological Separation Assay”) consistente en la separación inicial de las protínas de un complejo mediante SDS/PAGE, seguida de la transferencia de las proteínas de una banda específica a un tampón, donde se recaptura la proteína diana mediante una inmunoprecipitación convencional. Para demostrar la eficacia de ProCISA y su inocuidad sobre las modificaciones postraduccionales de las proteínas dianas, aislamos la proteína pp60Src o el receptor de IP3 de los complejos proteicos donde están ubicados, demostrando además en el caso de la primera, que ProCISA no altera el nivel de fosforilación y, por tanto, de activación de dicha proteína. Esta técnica podría tener además otras aplicaciones en el estudio de las modificaciones postraduccionales de las proteínas sin riesgo de obtener resultados erróneos por la contaminación en la muestra empleada con otras proteínas de similares características electroforéticas.
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Redondo, P.C., Rosado, J.A., Salido, G.M. et al. Protein complex immunological separation assay (ProCISA): a technique for investigating single protein properties. J. Physiol. Biochem. 64, 169–177 (2008). https://doi.org/10.1007/BF03178839
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DOI: https://doi.org/10.1007/BF03178839