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

可以使用FRAP、SPT和FRET等先进的显微技术来研究荧光标记蛋白的细胞动态。
在光漂白后荧光恢复（FRAP）实验中，使用聚焦的激光束照射含有目标蛋白的小区域，使荧光分子发生光漂白。
当标记的蛋白质扩散进入被漂白的区域后，该区域再次产生荧光。荧光恢复的速率决定了蛋白质的扩散速率。

单颗粒示踪技术（SPT）涉及使用与荧光染料偶联的抗体标记蛋白质。通过计算机增强的视频显微镜追踪单个蛋白质的运动。
荧光共振能量转移（FRET）用于检测两个带有荧光标记的蛋白质之间的接近程度。当两者距离超过十纳米时，不会发生荧光。如果蛋白质相互靠近，激发供体荧光基团将导致能量转移至受体荧光基团。
&#160;能量转移会降低供体的荧光，同时增强受体的发射，从而实现两者的可视化。

<ol>
	<li>Lippincott-Schwartz， J.、Snapp， EL 和 Phair， RD，2018 年。FRAP 的开发和增强作为研究蛋白质动力学的关键工具。生物物理学杂志 （Biophysical journal），第 115 卷第 7 期，第 1146-1155 页。</li>
	<li>Takanishi， CL， Bykova， EA， Cheng， W. 和 Zheng， J.，2006 年。活细胞中基于 GFP 的 FRET 分析。大脑研究，1091（1），第 132-139 页。</li>
	<li>Pantazis， A.， Westerberg， K.， Althoff， T.， Abramson， J. 和 Olcese， R.，2018 年。利用光诱导电子转移以光学方式确定蛋白质亚纳米级原子距离。自然通讯，9（1），第 1-12 页。</li>
</ol>

活细胞中的蛋白质动力学

Protein Dynamics in Living Cells

不同的基于荧光的技术用于研究活细胞中的蛋白质动力学。 这些技术包括 FRAP、FRET 和 PET。

光漂白后荧光恢复 (FRAP) 是一种基于荧光蛋白的检测技术，用于量化细胞内的蛋白质运动速率。这种方法将细胞的一小部分暴露在强激光束下。激光束会导致暴露区域中荧光基团标记的蛋白质发生永久性的光漂白。随着时间的推移，漂白的蛋白质扩散出去，而来自细胞其他部分的带有荧光标记的蛋白质会移动进来，使得该区域再次发出荧光。通过绘制相对荧光强度与所用时间的关系来量化运动速率，从而可以确定蛋白质在细胞内运动的速率。

福斯特共振能量转移 (FRET) 是一种用于确定两个蛋白质之间距离的分子技术。因此，FRET可以用作分子标尺。该技术使用两种荧光团标记的蛋白质，一种作为供体，另一种作为受体。在特定波长激发时，供体荧光团发射荧光能量，该能量被受体荧光团吸收。供体荧光团在能量转移后返回基态，而受体荧光团发出荧光，可通过荧光显微镜观察到。FRET 技术取决于三个因素：相互作用蛋白质之间的距离、供体和受体荧光团之间光谱重叠的程度以及能量转移过程中供体和受体荧光团的方向。仅当两个相互作用的蛋白质之间的距离为 10 nm 或更小时，正确定向的供体和受体荧光团之间的能量转移才会发生。

光诱导电子转移（PET）用于确定细胞中蛋白质之间的亚原子距离。在 PET 中，荧光基团吸收光并通过激发电子发射荧光信号。这个激发电子随后转移到受体上。在能量转移过程中，会发生氧化还原反应，从而在供体和受体蛋白质之间产生电荷分离。

不同的基于荧光的技术被用于研究活细胞中的蛋白质动态。这些技术包括 FRAP、FRET 和 PET。  
荧光……

Education

教育

JoVE Core

Cell Biology

细胞生物学

Unit 1

第一单元

Visualizing Cells, Tissues, and Molecules

细胞、组织与分子的可视化

KEY TERMS AND CONCEPTS

关键术语与概念

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

imaging biological samples with optical microscopy

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How Light Microscopes Bring Samples Into View

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...

phase contrast and differential interference contrast microscopy

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Microscope Contrast for Live, Unstained Cells

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fixation and sectioning

Microscope Slide Prep and Staining

Microscope slide preparation includes wet mounts, fixation, and staining. These steps help make specimens easier to see under a light microscope. They also help preserve cells and highlight important structures. A wet mount is the simplest slide preparation. The specimen is placed in a drop of liquid on the slide. A liquid sample can be added directly with a dropper, and a solid sample, such as a skin scraping, can be placed on the slide before the liquid is added. The liquid is sometimes...

immunofluorescence microscopy

Fluorescent Antibody Staining in Cells

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immunocytochemistry and immunohistochemistry

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Antibody Staining in Cells and Tissues

Immunocytochemistry (ICC) and immunohistochemistry (IHC) use antibodies to detect specific proteins or antigens in samples. ICC focuses on individual cells, such as blood cells and stem cells. IHC is used for tissue samples. These methods were first published by Albert Coons in 1941, when they were used to detect pneumococcal antigen in mouse tissue sections from mice infected with Pneumococcus. Both techniques can produce either a colored signal or a fluorescent signal. Enzyme-conjugated...

confocal fluorescence microscopy

Confocal Microscopy for Sharp Cell Images

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

total-internal-reflection-fluorescence-microscopy

Surface-Only Imaging with TIRF Microscopy

Total internal reflection fluorescence microscopy, or TIRF, is a microscope method used to view fluorophores near a solid surface with a higher refractive index, such as a glass coverslip. It is useful when scientists want to study signals close to the cell surface. TIRF works when light from a medium with a lower refractive index, such as air, hits the glass coverslip at a critical angle. Instead of entering the glass, the light undergoes total internal reflection. This reflection creates an...

atomic force microscopy

Atomic Force Microscopy for Surface Mapping

Atomic force microscopy is used to map the surface details of materials. It can examine ceramics, glass, polymers, and biological samples. The method gives far more resolution than an optical imaging system and produces three-dimensional surface profiles instead of flat 2D images. The probe is the key part of an AFM setup. It includes the cantilever and tip assembly, and it is also the part that is replaced most often. Constant contact with samples wears down the tip, so probe choice depends...

super-resolution fluorescence microscopy

Sharper Cell Images with Fluorescence Microscopy

Super-resolution fluorescence microscopy gives sharper images than standard fluorescence microscopy by reducing the point spread function, or PSF. The PSF is the light pattern from a point source that makes it look blurred. Because of this blur, each fluorescent spot appears larger than it really is. Nearby fluorophores can also overlap through their PSFs and make the image less clear. Several super-resolution methods have been developed to improve image detail. Photoactivated localization...

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Electron microscopy uses electrons instead of visible light to make images with much higher detail than a light microscope. Visible light limits the resolution of light microscopes, which usually magnify up to 1000X, and sometimes up to 1500X. Electrons have a much shorter wavelength, about 0.005 nm, so they can produce resolution as fine as 0.05 nm. An electron microscope can also magnify images up to 2,000,000X and reveal subcellular structures and some molecular structures, including single...

scanning electron microscopy

How SEM Reveals Surface Details

A scanning electron microscope (SEM) reveals the surface details of a sample with an electron beam. The beam scans across the sample in a two-dimensional pattern. SEM can image areas from about 1 centimeter down to 5 micrometers wide. SEM is useful for studying bacteria, viruses, tissues, and larger samples such as insects. Conventional SEM can produce magnification from 20X to 30,000X. It also offers spatial resolution of about 50 to 100 nanometers. The process starts when the electron gun...

transmission electron microscopy

How TEM Reveals Tiny Structures

Transmission electron microscopy uses electrons to reveal very small structures inside a specimen. The technique grew from the first electron microscope prototype developed in 1931 by physicist Ernst Ruska. He built on the idea that magnetic fields can direct an electron beam, just as lenses direct light in an optical microscope. In a transmission electron microscope, a hot tungsten element produces electrons. An electron gun then accelerates them with a potential difference, giving the beam...

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

Immunogold labeling in electron microscopy uses antibodies and tiny gold particles to find specific proteins in cells and tissues. The gold particles are very electron-dense, which means they show up clearly in electron microscope images. This gives researchers a high-resolution way to see where proteins are located and to compare their amounts under different stimulation conditions. The method can reveal proteins on the cell surface and inside cells. It has been used to study proteins...

cryo-electron microscopy

Cryo-EM for Native Biomolecule Imaging

Cryo-EM, or cryo-electron microscopy, lets scientists image biomolecules in their frozen, native state. It avoids the dehydration, fixation, and staining used in conventional electron microscopy, which can distort samples and create artifacts. It also reduces damage from the electron beam by using gentler imaging conditions. This method is useful when other structure tools have limits. X-ray diffraction needs a molecule to form a crystal, and that is not always possible. Even when...

electron microscope tomography and single-particle reconstruction

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3D Imaging with Electron Microscopy

Transmission electron microscopy (TEM) can build 3D images of biological samples. It does this with electron microscope tomography and single-particle reconstruction. Tomography can show cell parts or small cells in vivo, while single-particle reconstruction is used for macromolecules and macromolecular complexes in vitro only. Electron tomography can be done in TEM or in STEM, which stands for scanning transmission electron microscopy. STEM is mainly used for thick biological specimens. It...

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 Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

Fluorescent Methods for Protein Movement

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