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

Cellular dynamics of fluorescently labeled proteins can be studied using advanced microscopic techniques like FRAP, SPT, and FRET.
In fluorescence recovery after photobleaching or FRAP, a small region containing proteins of interest is irradiated with a focused laser beam to photobleach the fluorophores.
As labeled proteins diffuse into the bleached area, the region becomes fluorescent again. The rate of fluorescence recovery determines the diffusion rate of the proteins.

Single-particle tracking or SPT involves proteins tagged with fluorophore-bound antibodies. Individual protein movement is tracked using a computer-enhanced video microscope.
Forster resonance energy transfer or FRET measures the proximity of two fluorophore-tagged proteins. When the distance is more than ten nanometers, no fluorescence takes place. If the proteins move closer, exciting the donor fluorophore leads to energy transfer to the acceptor fluorophore.
&#160;The energy transfer reduces the dono&#39;s fluorescence while increasing the acceptor&#39;s emission, allowing their visualization.

eiwitdynamiek in levende cellen

Protein Dynamics in Living Cells

Er worden verschillende op fluorescentie gebaseerde technieken gebruikt om de eiwitdynamiek in levende cellen te bestuderen. Deze technieken omvatten FRAP, FRET en PET.

Fluorescerend herstel na fotobleken (FRAP) is een op fluorescerend eiwit gebaseerde detectietechniek die wordt gebruikt om de bewegingssnelheden van eiwitten in de cel te kwantificeren. Deze methode stelt een klein deel van de cel bloot aan een intense laserstraal. De laserstraal veroorzaakt permanente fotobleking van de met fluorofoor gemerkte eiwitten in het blootgestelde gebied. Terwijl de gebleekte eiwitten in de loop van de tijd naar buiten diffunderen en fluorescent gelabelde eiwitten uit andere delen van de cel naar binnen bewegen, wordt het gebied weer fluorescerend. De bewegingssnelheid wordt gekwantificeerd door de relatieve fluorescentie-intensiteit uit te zetten tegen de benodigde tijd, waardoor de snelheid kan worden bepaald waarmee eiwitten binnen de cel bewegen.

Förster-resonantie-energieoverdracht (FRET) is een moleculaire techniek die wordt gebruikt om de afstand tussen twee eiwitten te bepalen. FRET kan dus worden gebruikt als moleculaire liniaal. De techniek maakt gebruik van twee met fluorofoor gemerkte eiwitten, waarvan de ene fungeert als donor en de andere als acceptor. Bij excitatie bij een gespecificeerde golflengte zendt de donorfluorofoor fluorescentie-energie uit die wordt geabsorbeerd door de acceptorfluorofoor. De donorfluorofoor keert bij energieoverdracht terug naar de grondtoestand, terwijl de acceptorfluorofoor fluorescentie uitzendt die wordt gevisualiseerd met een fluorescentiemicroscoop. De FRET-techniek is afhankelijk van drie factoren: de afstand tussen interacterende eiwitten, de mate van spectrale overlap tussen de donor- en acceptor-fluoroforen, en de oriëntatie van de donor- en acceptor-fluorofoor tijdens energieoverdracht. De energieoverdracht tussen de correct georiënteerde donor- en acceptorfluoroforen kan alleen plaatsvinden als de afstand tussen de twee interacterende eiwitten 10 nm of minder bedraagt.

Foto-geïnduceerde elektronenoverdracht of PET bepaalt de subatomaire afstand tussen eiwitten in een cel. In PET absorbeert de fluorofoor het licht en zendt een fluorescentiesignaal uit via een aangeslagen elektron. Het aangeslagen elektron wordt overgebracht naar de receptor. Tijdens de energieoverdracht vindt een redoxreactie plaats, waardoor ladingsscheiding ontstaat tussen de donor- en acceptoreiwitten.

Eiwitdynamiek in levende cellen

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent ...

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JoVE Core

Cell Biology

Celbiologie

Unit 1

Eenheid 1

Visualizing Cells, Tissues, and Molecules

Visualisatie van cellen, weefsels en moleculen

KEY TERMS AND CONCEPTS

BELANGRIJKE TERMEN EN CONCEPTEN

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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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...

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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

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Fluorescent Methods for Protein Movement

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