Name: fluorescent methods for protein movement
Uploaded: 2023-04-30T12:00:21.000Z
Duration: 1 min 19 s
Channel: JoVE

La dinámica celular de las proteínas marcadas con fluorescencia se puede estudiar utilizando técnicas microscópicas avanzadas como FRAP, SPT y FRET.
En la recuperación de fluorescencia después del fotoblanqueo o FRAP, una pequeña región que contiene proteínas de interés se irradia con un rayo láser enfocado para fotoblanquear los fluoróforos.
A medida que las proteínas marcadas se difunden en el área blanqueada, la región vuelve a ser fluorescente. La tasa de recuperación de fluorescencia determina la velocidad de difusión de las proteínas.

El seguimiento de una sola partícula o SPT involucra proteínas marcadas con anticuerpos unidos a fluoróforos. El movimiento individual de las proteínas se rastrea utilizando un microscopio de video mejorado por computadora.
La transferencia de energía por resonancia de Forster o FRET mide la proximidad de dos proteínas marcadas con fluoróforos. Cuando la distancia es superior a diez nanómetros, no se produce fluorescencia. Si las proteínas se acercan, la excitación del fluoróforo donante conduce a la transferencia de energía al fluoróforo aceptor.
 La transferencia de energía reduce la fluorescencia del donante a la vez que aumenta la emisión del aceptor, permitiendo su visualización.

<ol>
	<li>Lippincott-Schwartz, J., Snapp, E.L. y Phair, R.D., 2018. El desarrollo y mejora de FRAP como una herramienta clave para investigar la dinámica de proteínas. Revista de Biofísica, 115(7), pp.1146-1155.</li>
	<li>Takanishi, C.L., Bykova, E.A., Cheng, W. y Zheng, J., 2006. Análisis FRET basado en GFP en células vivas. Investigación del cerebro, 1091(1), pp.132-139.</li>
	<li>Pantazis, A., Westerberg, K., Althoff, T., Abramson, J. y Olcese, R., 2018. Aprovechamiento de la transferencia de electrones fotoinducida para determinar ópticamente las distancias atómicas a subnanoescala de las proteínas. Comunicaciones de la Naturaleza, 9(1), pp.1-12.</li>
</ol>

dinámica de proteínas en células vivas

Protein Dynamics in Living Cells

Se utilizan diferentes técnicas basadas en fluorescencia para estudiar la dinámica de las proteínas en células vivas. Estas técnicas incluyen FRAP, FRET y PET.

La recuperación fluorescente después del fotoblanqueo (FRAP) es una técnica de detección basada en proteínas fluorescentes que se utiliza para cuantificar las tasas de movimiento de proteínas dentro de la célula. Este método expone una pequeña porción de la célula a un intenso rayo láser. El rayo láser provoca un fotoblanqueo permanente de las proteínas marcadas con fluoróforos en la región expuesta. A medida que las proteínas blanqueadas se difunden con el tiempo y las proteínas marcadas con fluorescencia de otras partes de la célula entran, la región vuelve a ser fluorescente. La velocidad de movimiento se cuantifica trazando la intensidad relativa de fluorescencia frente al tiempo necesario, lo que permite determinar la velocidad a la que se mueve la proteína dentro de la célula.

La transferencia de energía por resonancia de Förster (FRET) es una técnica molecular utilizada para determinar la distancia entre dos proteínas. Por tanto, FRET se puede utilizar como regla molecular. La técnica utiliza dos proteínas marcadas con fluoróforos, una que actúa como donante y la otra como aceptora. Tras la excitación a una longitud de onda específica, el fluoróforo donante emite energía de fluorescencia que es absorbida por el fluoróforo aceptor. El fluoróforo donante vuelve al estado fundamental tras la transferencia de energía, mientras que el fluoróforo aceptor emite fluorescencia que se visualiza con un microscopio de fluorescencia. La técnica FRET depende de tres factores: la distancia entre las proteínas que interactúan, el grado de superposición espectral entre los fluoróforos donante y aceptor, y la orientación del fluoróforo donante y aceptor durante la transferencia de energía. La transferencia de energía entre los fluoróforos donante y aceptor correctamente orientados sólo puede ocurrir cuando la distancia entre las dos proteínas que interactúan es de 10 nm o menos.

La transferencia de electrones fotoinducida o PET determina la distancia subatómica entre las proteínas de una célula. En PET, el fluoróforo absorbe la luz y emite una señal de fluorescencia a través de un electrón excitado. El electrón excitado se transfiere al receptor. Durante la transferencia de energía se produce una reacción redox, generando separación de carga entre las proteínas donadoras y aceptoras.

Dinámica de proteínas en células vivas

Se utilizan diferentes técnicas basadas en fluorescencia para estudiar la dinámica de las proteínas en las células vivas. Estas técnicas incluyen FRAP, FRET y PET.
Fluorescente...

Education

Educación

JoVE Core

Cell Biology

Biología Celular

Unit 1

Unidad 1

Visualizing Cells, Tissues, and Molecules

Visualización de células, tejidos y moléculas

KEY TERMS AND CONCEPTS

TÉRMINOS Y CONCEPTOS CLAVE

fluorescent methods for protein movement

Protein movement in living cells can be measured with fluorescence-based methods such as FRAP, FRET, and PET. These techniques help scientists track how proteins move, interact, and change distance inside the cell.

Fluorescent recovery after photobleaching, or FRAP, measures protein movement rates within the cell. In this method, a small region is exposed to a strong laser beam. The beam permanently bleaches fluorophore-tagged proteins in that area. Over time, bleached proteins diffuse away and fluorescent proteins from other parts of the cell move in. The region then becomes fluorescent again, and the change in intensity is plotted against time to calculate how fast the protein moves.

Fӧrster resonance energy transfer, or FRET, measures the distance between two proteins and can act like a molecular ruler. It uses two fluorophore-tagged proteins, a donor and an acceptor. When the donor is excited at a specific wavelength, it transfers fluorescence energy to the acceptor. The donor returns to the ground state, and the acceptor emits fluorescence that can be seen with a fluorescence microscope. FRET depends on distance between the proteins, spectral overlap between the fluorophores, and their orientation during energy transfer. This transfer occurs only when the interacting proteins are 10 nm apart or less.

Photoinduced electron transfer, or PET, measures sub-atomic distance between proteins in a cell. In PET, the fluorophore absorbs light and sends out a fluorescence signal through an excited electron. That electron is transferred to a receptor. During this transfer, a redox reaction creates charge separation between the donor and acceptor proteins.

Fluorescent Methods for Protein Movement

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Optical microscopy uses light and lenses to make tiny biological samples visible. Antonie van Leeuwenhoek built the first compound optical microscope in the 17th century to view blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister developed an essentially modern light microscope. During the 20th century, microscopes gained better magnification and resolution. A sample is placed on a glass slide and clipped onto the stage, which is the flat platform of the microscope. The...

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confocal fluorescence microscopy

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Confocal microscopy is an advanced imaging method used to capture sharp fluorescence images. Its main advantage is that it blocks out-of-focus light from the sample with pinholes. That helps produce high-resolution images with strong contrast. Unlike a standard optical microscope, a confocal microscope uses a focused laser beam to scan the sample surface at different z-planes. This makes it especially useful for thick specimens, such as biofilms. These samples can often be examined alive and...

protein dynamics in living cells

Protein movement in living cells can be measured with fluorescence-based methods such as FRAP, FRET, and PET. These techniques help scientists track how proteins move, interact, and change distance inside the cell. Fluorescent recovery after photobleaching, or FRAP, measures protein movement rates within the cell. In this method, a small region is exposed to a strong laser beam. The beam permanently bleaches fluorophore-tagged proteins in that area. Over time, bleached proteins diffuse away...

total internal reflection fluorescence microscopy

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Surface-Only Imaging with TIRF Microscopy

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scanning electron microscopy

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transmission electron microscopy

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preparation of samples for electron microscopy

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Electron Microscopy Sample Prep Methods

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immunogold electron microscopy

Immunogold Labeling in Electron Microscopy

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cryo-electron microscopy

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3D Imaging with Electron Microscopy

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key terms and concepts

Learn how FRAP, FRET, and PET use fluorescence to measure protein movement, protein distance, and protein interactions in living cells.

Fluorescent Methods for Protein Movement | JoVE K12

cell biology

JoVE Core Biología Celular ilustra procesos celulares complejos mediante completos vídeos animados fáciles de entender. Esta colección se centra en los componentes esenciales de las células y su funcionamiento, incluyendo los orgánulos y las membranas, el transporte celular y el metabolismo energético celular. Todo este material se complementa con los vídeos de científicos en acción, que muestran la aplicación de la biología celular en experimentos reales de investigación.

Fluorescent Methods for Protein Movement

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