RESUMEN
Electron microscopy (EM) is a rapidly growing area of structural biology that permits us to decode biological assemblies at the nanoscale. To examine biological materials for single particle EM analysis, purified assemblies must be obtained using biochemical separation techniques. Here, we describe effective methodologies for isolating histidine (his)-tagged protein assemblies from the nucleus of disease-relevant cell lines. We further demonstrate how isolated assemblies are visualized using single particle EM techniques and provide representative results for each step in the process.
Asunto(s)
Microscopía Electrónica/métodos , Receptores de Superficie Celular/química , Imagen Individual de Molécula/métodos , Métodos Analíticos de la Preparación de la Muestra , Animales , Línea Celular , Cromatografía de Afinidad , Descubrimiento de Drogas , Histidina/química , Humanos , Ratones , Modelos Moleculares , Orgánulos/ultraestructura , Compuestos Organometálicos/químicaRESUMEN
Understanding the properties of protein-based therapeutics is a common goal of biologists and physicians. Technical barriers in the direct observation of small proteins or therapeutic agents can limit our knowledge of how they function in solution and in the body. Electron microscopy (EM) imaging performed in a liquid environment permits us to peer into the active world of cells and molecules at the nanoscale. Here, we employ liquid cell EM to directly visualize a protein-based therapeutic in its native conformation and aggregate state in a time-resolved manner. In combination with quantitative analyses, information from this work contributes new molecular insights toward understanding the behaviours of immunotherapies in a solution state that mimics the human body.
Asunto(s)
Microscopía Electrónica/métodos , Agregado de Proteínas , Composición de Medicamentos , Interferón-alfa/química , Interferón-alfa/uso terapéutico , Polietilenglicoles/química , Conformación Proteica , Factores de TiempoRESUMEN
Currently, there remains a critical need to develop real-time imaging resources for life sciences. Here, we demonstrate the use of high resolution in situ imaging to observe biological complexes in liquid at the nanoscale. Using a model virus system, we produced the first time-resolved videos of individual biological complexes moving in solution within an electron microscope.