La determinación de los Planes de unión de hielo de Anticongelante Proteínas por Basado en la fluorescencia de Hielo Plane Affinity

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Basu, K., Garnham, C. P., Nishimiya, Y., Tsuda, S., Braslavsky, I., Davies, P. Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity. J. Vis. Exp. (83), e51185, doi:10.3791/51185 (2014).

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Abstract

Proteínas anticongelantes (AFPs) se expresan en una variedad de organismos resistentes al frío para prevenir o retardar el crecimiento del hielo interior. AFP se unen a los planos específicos de hielo a través de sus superficies de unión de hielo. Afinidad plano de hielo a base de fluorescencia (FIPA) análisis es una técnica modificada se utiliza para determinar los planos de hielo a la que se unen la AFP. FIPA se basa en el método de hielo-aguafuerte original para la determinación de hielo-planos limitados-AFP. Produce imágenes más claras en una época experimental acortado. En el análisis del FIPA, AFP están marcados con fluorescencia con una etiqueta quimérico o un colorante covalente a continuación, incorporado lentamente en un solo cristal de hielo macroscópica, que ha sido preformado en un hemisferio y orientado para determinar los-y C-ejes. El hemisferio hielo con destino a la AFP se crea una imagen con luz UV para visualizar planos con destino a la AFP el uso de filtros para bloquear la luz no específica. Etiquetado fluorescente de las AFPs permite la monitorización en tiempo real de AFP adsorción en hielo. Se han encontrado los etiquetasno influir en los planos a los que se unen las AFP. Análisis FIPA también introduce la posibilidad de obligar a más de un etiquetado diferente AFP en el mismo solo cristal de hielo para ayudar a diferenciar sus planos vinculantes. Estas aplicaciones de FIPA están ayudando a avanzar en nuestra comprensión de cómo las AFP se unen a hielo para detener su crecimiento y por qué muchos organismos AFP productores expresan múltiples isoformas de AFP.

Materials

Name Company Catalog Number Comments
NESLAB RTE Refrigerating Bath/Circulators Thermo Scientific RTE7
Ethylene glycol, Premixed Antifreeze/Coolant Certified 29-3037-0 Common automotive antifreeze
Cold finger not available not available Custom made with brass (9 cm long, 1.5 cm outer diameter)
Hemispherical cup not available not available Custom made with resin (8 cm outer diameter, 6 cm inner diameter)
High Dual Output Lighting System Lightools Research LT-99D2, Illumatools DLS 120 volts AC, LT-9470FX, LT-9549FX Additional and custom excitation filters can be purchased from Lightools Research
Camera Canon EOS 50D
Emission Filters Lightools Research LT-9EFPVG, LT-9GFPVG, LT-9RFPVG Filter ring adapter may be required to fit filter onto camera lens

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