生物物理学


分类

现刊
0 Q&A 52 Views Jun 5, 2023

Lipid-conjugated pH sensors based on fluorophores coupled to lipids are a powerful tool for monitoring pH gradients in biological microcompartments and reconstituted membrane systems. This protocol describes the synthesis of pH sensors based on amine-reactive pHrodo esters and the amino phospholipid phosphatidylethanolamine. The major features of this sensor include efficient partitioning into membranes and strong fluorescence under acidic conditions. The protocol described here can be used as a template to couple other amine-reactive fluorophores to phosphatidylethanolamines.


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往期刊物
0 Q&A 255 Views May 20, 2023

ATPase assays are a common tool for the characterization of purified ATPases. Here, we describe a radioactive [γ-32P]-ATP-based approach, utilizing complex formation with molybdate for phase separation of the free phosphate from non-hydrolyzed, intact ATP. The high sensitivity of this assay, compared to common assays such as the Malachite green or NADH-coupled assay, enables the examination of proteins with low ATPase activity or low purification yields. This assay can be used on purified proteins for several applications including the identification of substrates, determination of the effect of mutations on ATPase activity, and testing specific ATPase inhibitors. Furthermore, the protocol outlined here can be adapted to measure the activity of reconstituted ATPases.


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0 Q&A 335 Views Apr 20, 2023

In cells, p62/SQSTM1 undergoes liquid–liquid phase separation (LLPS) with poly-ubiquitin chains to form p62 bodies that work as a hub for various cellular events, including selective autophagy. Cytoskeleton components such as Arp2/3-derived branched actin network and motor protein myosin 1D have been shown to actively participate in the formation of phase-separated p62 bodies. Here, we describe a detailed protocol on the purification of p62 and other proteins, the assembly of the branched actin network, and the reconstitution of p62 bodies along with cytoskeletal structures in vitro. This cell-free reconstitution of p62 bodies vividly mimics the phenomenon in which low concentrations of protein in vivo rely on cytoskeleton dynamics to increase the local concentration to reach the threshold for phase separation. This protocol provides an easily implemented and typical model system to study cytoskeleton-involved protein phase separation.

0 Q&A 274 Views Mar 5, 2023

Lipid droplets (LD), triglycerides and sterol esters among them, are well known for their capacity as lipid storage organelles. Recently, they have emerged as critical cytoplasmic structures involved in numerous biological functions. LD storage is generated de novo by the cell and provides an energy reserve, lipid precursors, and cell protection. Moreover, LD accumulation can be observed in some pathologies as obesity, atherosclerosis, or lung diseases. Fluorescence imaging techniques are the most widely used techniques to visualize cellular compartments in live cells, including LD. Nevertheless, presence of fluorophores can damage subcellular components and induce cytotoxicity, or even alter the dynamics of the organelles. As an alternative to fluorescence microscopy, label-free techniques such as stimulated Raman scattering and coherent anti-stokes Raman scattering microscopy offer a solution to avoid the undesirable effects caused by dyes and fluorescent proteins, but are expensive and complex. Here, we describe a label-free method using live-cell imaging by 3D holotomographic microscopy (Nanolive) to visualize LD accumulation in the MH-S alveolar macrophage cell line after treatment with oleic acid, a monounsaturated fatty acid that promotes lipid accumulation.

0 Q&A 487 Views Feb 20, 2023

The cell surfaceome is of vital importance across physiology, developmental biology, and disease states alike. The precise identification of proteins and their regulatory mechanisms at the cell membrane has been challenging and is typically determined using confocal microscopy, two-photon microscopy, or total internal reflection fluorescence microscopy (TIRFM). Of these, TIRFM is the most precise, as it harnesses the generation of a spatially delimited evanescent wave at the interface of two surfaces with distinct refractive indices. The limited penetration of the evanescent wave illuminates a narrow specimen field, which facilitates the localization of fluorescently tagged proteins at the cell membrane but not inside of the cell. In addition to constraining the depth of the image, TIRFM also significantly enhances the signal-to-noise ratio, which is particularly valuable in the study of live cells. Here, we detail a protocol for micromirror TIRFM analysis of optogenetically activated protein kinase C-ϵ in HEK293-T cells, as well as data analysis to demonstrate the translocation of this construct to the cell-surface following optogenetic activation.


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0 Q&A 720 Views Jan 20, 2023

Single-particle electron cryo-microscopy (cryo-EM) is an effective tool to determine high-resolution structures of macromolecular complexes. Its lower requirements for sample concentration and purity make it an accessible method to determine structures of low-abundant protein complexes, such as those isolated from native sources. While there are many approaches to protein purification for cryo-EM, attaining suitable particle quality and abundance is generally the major bottleneck to the typical single-particle project workflow. Here, we present a protocol using budding yeast (S. cerevisiae), in which a tractable immunoprecipitation tag (3xFLAG) is appended at the endogenous locus of a gene of interest (GOI). The modified gene is expressed under its endogenous promoter, and cells are grown and harvested using standard procedures. Our protocol describes the steps in which the tagged proteins and their associated complexes are isolated within three hours of thawing cell lysates, after which the recovered proteins are used directly for cryo-EM specimen preparation. The prioritization of speed maximizes the ability to recover intact, scarce complexes. The protocol is generalizable to soluble yeast proteins that tolerate C-terminal epitope tags.


Graphical abstract




Overview of lysate-to-grid workflow. Yeast cells are transformed to express a tractable tag on a gene of interest. Following cell culture and lysis, particles of interest are rapidly isolated by co-immunoprecipitation and prepared for cryo-EM imaging (created with BioRender.com).

0 Q&A 632 Views Dec 5, 2022

Cryo-electron tomography (cryo-ET) is a formidable technique to observe the inner workings of vitrified cells at a nanometric resolution in near-native conditions and in three-dimensions. One consequent drawback of this technique is the sample thickness, for two reasons: i) achieving proper vitrification of the sample gets increasingly difficult with sample thickness, and ii) cryo-ET relies on transmission electron microscopy (TEM), requiring thin samples for proper electron transmittance (<500 nm). For samples exceeding this thickness limit, thinning methods can be used to render the sample amenable for cryo-ET. Cryo-focused ion beam (cryo-FIB) milling is one of them and despite having hugely benefitted the fields of animal cell biology, virology, microbiology, and even crystallography, plant cells are still virtually unexplored by cryo-ET, in particular because they are generally orders of magnitude bigger than bacteria, viruses, or animal cells (at least 10 μm thick) and difficult to process by cryo-FIB milling. Here, we detail a preparation method where abaxial epidermal onion cell wall peels are separated from the epidermal cells and subsequently plunge frozen, cryo-FIB milled, and screened by cryo-ET in order to acquire high resolution tomographic data for analyzing the organization of the cell wall.

0 Q&A 1059 Views Nov 20, 2022

The study and use of decellularized extracellular matrix (dECM) in tissue engineering, regenerative medicine, and pathophysiology have become more prevalent in recent years. To obtain dECM, numerous decellularization procedures have been developed for the entire organ or tissue blocks, employing either perfusion of decellularizing agents through the tissue’s vessels or submersion of large sections in decellularizing solutions. However, none of these protocols are suitable for thin tissue slices (less than 100 µm) or allow side-by-side analysis of native and dECM consecutive tissue slices. Here, we present a detailed protocol to decellularize tissue sections while maintaining the sample attached to a glass slide. This protocol consists of consecutive washes and incubations of simple decellularizing agents: ultrapure water, sodium deoxycholate (SD) 2%, and deoxyribonuclease I solution 0.3 mg/mL (DNase I). This novel method has been optimized for a faster decellularization time (2–3 h) and a better correlation between dECM properties and native tissue-specific biomarkers, and has been tested in different types of tissues and species, obtaining similar results. Furthermore, this method can be used for scarce and valuable samples such as clinical biopsies.

0 Q&A 1509 Views Nov 5, 2022

Cryo-focused ion beam (FIB) milling of vitrified specimens is emerging as a powerful method for in situ specimen preparation. It allows for the preservation of native and near-native conditions in cells, and can reveal the molecular structure of protein complexes when combined with cryo-electron tomography (cryo-ET) and sub-tomogram averaging. Cryo-FIB milling is often performed on plunge-frozen specimens of limited thickness. However, this approach may have several disadvantages, including low throughput for cells that are small, or at low concentration, or poorly distributed across accessible areas of the grid, as well as for samples that may adopt a preferred orientation. Here, we present a detailed description of the “Waffle Method” protocol for vitrifying thick specimens followed by a semi-automated milling procedure using the Thermo Fisher Scientific (TFS) Aquilos 2 cryo-FIB/scanning electron microscope (SEM) instrument and AutoTEM Cryo software to produce cryo-lamellae. With this protocol, cryo-lamellae may be generated from specimens, such as microsporidia spores, yeast, bacteria, and mammalian cells, as well as purified proteins and protein complexes. An experienced lab can perform the entire protocol presented here within an 8-hour working day, resulting in two to three cryo-lamellae with target thicknesses of 100–200 nm and dimensions of approximately 12 μm width and 15–20 μm length. For cryo-FIB/SEMs with particularly low-contamination chambers, the protocol can be extended to overnight milling, resulting in up to 16 cryo-lamellae in 24 h.


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0 Q&A 825 Views Nov 5, 2022

Aptamers have been selected with strong affinity and high selectivity for a wide range of targets, as recently highlighted by the development of aptamer-based sensors that can differentiate infectious from non-infectious viruses, including human adenovirus and SARS-CoV-2. Accurate determination of the binding affinity between the DNA aptamers and their viral targets is the first step to understanding the molecular recognition of viral particles and the potential uses of aptamers in various diagnostics and therapeutic applications. Here, we describe protocols to obtain the binding curve of the DNA aptamers to SARS-CoV-2 using Enzyme-Linked Oligonucleotide Assay (ELONA) and MicroScale Thermophoresis (MST). These methods allow for the determination of the binding affinity of the aptamer to the infectious SARS-CoV-2 and the selectivity of this aptamer against the same SARS-CoV-2 that has been rendered non-infectious by UV inactivation, and other viruses. Compared to other techniques like Electrophoretic Mobility Shift Assay (EMSA), Surface Plasmon Resonance (SPR), and Isothermal Titration Calorimetry (ITC), these methods have advantages for working with larger particles like viruses and with samples that require biosafety level 2 facilities.

0 Q&A 645 Views Nov 5, 2022

Reconstitution of membrane proteins into large unilamellar vesicles is an essential approach for their functional analysis under chemically defined conditions. The orientation of the protein in the liposomal membrane after reconstitution depends on many parameters, and its assessment is important prior to functional measurements. Common approaches for determining the orientation of a membrane-inserted protein are based on limited proteolytic digest, impermeable labeling reagents for specific amino acids, or membrane-impermeable quenchers for fluorescent proteins. Here, we describe a simple site-specific fluorescent assay based on self-labeling enzyme tags to determine the orientation of membrane proteins after reconstitution, exemplified on a reconstituted SNAP-tag plant H+-ATPase. This versatile method should benefit the optimization of reconstitution conditions and the analysis of many types of membrane proteins.


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