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

형광 표지 단백질의 세포 역학은 FRAP, SPT 및 FRET와 같은 고급 현미경 기술을 사용하여 연구할 수 있습니다.
광표백 또는 FRAP 후 형광 회복에서는 관심 단백질이 포함된 작은 영역에 집속 레이저 빔을 조사하여 형광단을 광표백합니다.
표지된 단백질이 표백된 영역으로 확산되면 해당 영역이 다시 형광이 됩니다. 형광 회수율은 단백질의 확산 속도를 결정합니다.

단일 입자 추적 또는 SPT는 형광단 결합 항체로 태그된 단백질을 포함합니다. 개별 단백질의 움직임은 컴퓨터로 강화된 비디오 현미경을 사용하여 추적됩니다.
Forster 공명 에너지 전달 또는 FRET는 두 개의 형광단 표지 단백질의 근접성을 측정합니다. 거리가 10나노미터 이상이면 형광이 발생하지 않습니다. 단백질이 더 가까이 이동하면 공여체 형광단을 여기시켜 수용체 형광단으로 에너지를 전달합니다.
 에너지 전달은 도노의 형광을 감소시키는 동시에 수용체의 방출을 증가시켜 시각화를 가능하게 합니다.

<ol>
	<li>Lippincott-Schwartz, J., Snapp, EL 및 Phair, RD, 2018. 단백질 역학을 조사하기 위한 핵심 도구로서의 FRAP의 개발 및 향상. 생물물리학 저널, 115(7), pp.1146-1155.</li>
	<li>Takanishi, C.L., Bykova, E.A., Cheng, W. 및 Zheng, J., 2006년. 살아있는 세포에서 GFP 기반 FRET 분석. 뇌연구, 1091(1), pp.132-139.</li>
	<li>Pantazis, A., Westerberg, K., Althoff, T., Abramson, J. 및 Olcese, R., 2018. 광유도 전자 전달을 활용하여 단백질 나노 미만의 원자 거리를 광학적으로 결정합니다. 네이처 커뮤니케이션, 9(1), pp.1-12.</li>
</ol>

살아있는 세포의 단백질 역학

Protein Dynamics in Living Cells

살아있는 세포의 단백질 역학을 연구하기 위해 다양한 형광 기반 기술이 사용됩니다. 이러한 기술에는 FRAP, FRET 및 PET가 포함됩니다.

광표백 후 형광 회복(FRAP)은 세포 내 단백질 이동 속도를 정량화하는 데 사용되는 형광 단백질 기반 검출 기술입니다. 이 방법은 세포의 작은 부분을 강렬한 레이저 빔에 노출시킵니다. 레이저 빔은 노출된 영역에서 형광단 태그가 붙은 단백질의 영구적인 광표백을 유발합니다. 시간이 지남에 따라 표백된 단백질이 확산되고 세포의 다른 부분에서 형광 표지된 단백질이 이동함에 따라 해당 영역은 다시 형광이 됩니다. 이동 속도는 상대 형광 강도 대 소요 시간을 플롯하여 정량화되며, 이를 통해 세포 내에서 단백질이 이동하는 속도를 결정할 수 있습니다.

포스터 에너지전이(FRET)은 두 단백질 사이의 거리를 결정하는 데 사용되는 분자 기술입니다. 따라서 FRET는 분자 눈금자로 사용될 수 있습니다. 이 기술은 두 개의 형광단 태그가 붙은 단백질을 사용하는데, 하나는 기증자 역할을 하고 다른 하나는 수용체 역할을 합니다. 특정 파장에서 여기되면 도너 형광단은 수용체 형광단에 의해 흡수되는 형광 에너지를 방출합니다. 기증자 형광단은 에너지 전달 시 바닥 상태로 돌아가는 반면 수용체 형광단은 형광 현미경으로 시각화되는 형광을 방출합니다. FRET 기술은 상호 작용하는 단백질 사이의 거리, 공여자와 수용자 형광단 사이의 스펙트럼 중첩 정도, 에너지 전달 중 공여자와 수용자 형광단의 방향 등 세 가지 요소에 따라 달라집니다. 올바른 방향의 기증자와 수용체 형광단 사이의 에너지 전달은 상호 작용하는 두 단백질 사이의 거리가 10 nm 이하인 경우에만 발생할 수 있습니다.

광유발 전자 전달(PET)은 세포 내 단백질 사이의 아원자 거리를 결정합니다. PET에서 형광단은 빛을 흡수하고 여기된 전자를 통해 형광 신호를 방출합니다. 여기된 전자는 수용체로 전달됩니다. 에너지 전달 중에 산화환원 반응이 일어나 공여체와 수용체 단백질 사이에 전하 분리가 발생합니다.

살아있는 세포의 단백질 역학을 연구하기 위해 다양한 형광 기반 기술이 사용됩니다. 이러한 기술에는 FRAP, FRET 및 PET가 포함됩니다.
형광...

Education

교육

JoVE Core

Cell Biology

세포생물학

Unit 1

제1단원

Visualizing Cells, Tissues, and Molecules

세포, 조직 및 분자의 시각화

KEY TERMS AND CONCEPTS

주요 용어 및 개념

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

imaging-biological-samples-with-optical-microscopy

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

phase-contrast-and-differential-interference-contrast-microscopy

Microscope Contrast for Live, Unstained Cells

Phase-contrast and differential interference contrast microscopy help scientists see live cells without stains. Both methods boost contrast so thin, transparent structures become easier to study under the microscope. Phase-contrast microscopes use interference between light that passes straight through a cell and light that is refracted by cell parts. The microscope uses an annular stop in the condenser to make a hollow cone of light. The objective lens also contains a phase plate with a phase...

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

Fluorescent antibody staining uses a fluorescence microscope to make cells and microbes visible in bright colors against a dark background. The microscope works with fluorochromes, which are fluorescent dyes that absorb energy from a light source and then give off visible light. Some fluorochromes are natural, such as chlorophylls. Others are added to the specimen to improve contrast. Common examples of fluorochromes include Texas red and FITC. The method can also use nucleic acid dyes such as...

immunocytochemistry and immunohistochemistry

immunocytochemistry-and-immunohistochemistry

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...

overview of electron microscopy

Electron Microscopy: TEM vs SEM

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

preparation-of-samples-for-electron-microscopy

Electron Microscopy Sample Prep Methods

Electron microscopy sample prep methods help biological samples survive imaging in TEM and SEM. Before a specimen can be viewed, it must be fixed, or stabilized, so the electron beam does not damage it. It also must be dried well, or desiccated, because the microscope uses a vacuum. Fixation should happen as quickly as possible. Once a sample leaves its natural environment, its properties begin to change. In tissue samples, oxygen levels can drop and change the appearance of mitochondria. That...

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

electron-microscope-tomography-and-single-particle-reconstruction

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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