1。样蛋白的制备<ol><li>在500μL的离心管中，结合以下内容： </li></ol><table border="1"><tbody><tr><td>试剂</td><td>卷补充： </td></tr><tr><td> *蛋白质样品（1毫克） </td><td>至112μL </td></tr><tr><td> 在墨盒套件提供的样品缓冲液（5X ） </td><td> 30μL </td></tr><tr><td>数码地面电视1M </td><td> 8μL </td></tr><tr><td> 18ΩH 2 O </td><td> x微升到总样本量150μL** </td></tr></tbody></table><ol start="2"><li>变性的样品在95℃5分钟。冷却至室温。 </li></ol> *要分开的蛋白质的总金额将取决于样品的复杂性。 对于更复杂的样品，如组织匀浆，建议150-200微克每通道 。 **如果样品体积小于112μL，加18ΩH 2 O，使最终体积为150μL。 2。载入GELFREE盒<ol><li>删除GELFREE 8100盒，铝箔袋。 </li><li>删除和丢弃的板盖。 </li><li>使用吸液管从墨盒车厢中取出的存储缓冲区。如果墨盒的所有8个商会将被使用，墨盒可以倒排从车厢内的存储缓冲区。 </li><li>墨盒准备要加载的GELFREE运行缓冲液中加入每个墨盒商会如下： </li></ol><table border="1"><tbody><tr><td> GELFREE墨盒会所</td><td> GELFREE运行缓冲液中添加量</td></tr><tr><td>阳极缓冲水库</td><td> 8毫升</td></tr><tr><td>阴极缓冲水库</td><td> 6毫升</td></tr><tr><td>收集商会</td><td>各加入100μL </td></tr></tbody></table><ol start="5"><li>取下并丢弃任何缓冲区从样品装载室。 </li><li> 150μL的样品装入加载使用一个8通道移液器的商会。 </li><li>装入墨盒放入GELFREE 8100分馏站。降低电极阵列，并盖上盖子。 </li></ol> 3。运行墨盒<ol><li>使用该仪器的触摸屏显示，按方法 “按钮，以查看预编程的方法列表。预编程的方法组成，每个设置的电压和时间为基础的，旨在解决一个特定范围的分子量内的蛋白质暂停。 GELFREE 8100会自动暂停馏分收集方法在指定的时间间隔。 </li><li>一旦适当的方法已经确定，按检索按钮，并输入所需的预编程的方法使用屏幕上的键盘。按“OK”，然后按Apply按钮。该方法已应用于将出现在主屏幕的底部。 </li><li>要选择的渠道（样本数），将用于在运行时，按主屏幕上的频道按钮。要选择所有八个通道，按全部选择。然后，按返回到主屏幕。 </li><li>确保安全盖被关闭，并且在屏幕底部的指示灯是绿色的。 </li><li>要开始的蛋白质分离，按开始 。分馏站将自动暂停，当它的时间来收集馏分注意：当系统运行时，显示在屏幕左侧的框中的圆圈描绘每个通道的状态。 绿色圆圈表示的通道被激活，一个黄色的圆圈表示，该仪器已暂停，和一个红色圆圈表示该通道已失败。适用的电压和电流显示通道状态界的权利。 分数只应删除时的状态界变为黄色和仪器的屏幕上出现一个文本警报。 </li><li>要删除分数，打开仪器盖，并使用一个8通道移液器撤回150μL每个收集室。 </li><li>一旦分数已被删除，用GELFREE运行缓冲液收集室两次，加入100μL，每通道，吹打起来，上下两次。 </li><li>洗涤后，加入100uL GELFREE运行到缓冲区回来的收集室，盖上盖子，按下继续按钮。 </li><li> GELFREE 8100将运行，直到下一次的时间间隔。在实验中剩余的总时间显示在屏幕的右侧。 剩余时间之间切换，时间过去了，暂停时间，按下按钮，上面显示的时间框。 </li><li>一旦所有的分数已经合作llected，他们可以立即被用于进一步的下游制剂，如等电聚焦电泳，或使用液相色谱质谱或免疫分析。 </li></ol> 4。代表性的成果被分成12分数每一个牛的肝组织匀浆200微克，使用GELFREE分馏系统。 6μL每个分数上运行一个标准的一维SDS - PAGE凝胶银染。如图1所示，蛋白质样品分割成12个不同的分数，跨越分子量范围从3.5kDa 150kDa。分子量标准（ColorBurst，Sigma公司）也显示在1和14道。此处所示的凝胶，强调通过对蛋白质的分离GELFREE 8100系统的高回收率。此处所示的凝胶代表从200微克的蛋白质的4％。 <img src="/files/ftp_upload/1842/1842fig1tmb.jpg" alt="图1" border="0" /> 图1。从牛的肝脏蛋白质匀浆分开使用GELFREE分馏系统 ，6μL每150μL的分数被加载和运行上的一维SDS PAGE凝胶电泳和银染。还装载了一个分子量（MW）的标准表示。请<a href="https://www.jove.com/files/ftp_upload/1842/1842fig1.jpg">点击这里</a>看到图1的放大版本。

<ol>
	<li>Tran, J. C., Doucette, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Size+Separation+of+Intact+Proteins+in+Solution+Phase+for+Mass+Spectrometry.">Multiplexed Size Separation of Intact Proteins in Solution Phase for Mass Spectrometry.</a> Anal Chem.. , (2009).</li></ol>

The authors of this article are employed by Protein Discovery that produces reagents and instruments used in this article.

GELFREE 8100分馏系统，提供了一种快速，复杂的蛋白质样品分割成离散分子量为基础的分数简单，重复性好方法。这新颖的分馏方法，消除了乐队或现场切割的需要，有可能孤立广泛的群众范围，在90分钟内的蛋白质。负载能力相对大量的样品和恢复液相分数，这是典型的更难以察觉的低丰度蛋白质的分离和分析，特别是在有用的。由此产生的分数可以被用来在广泛的应用，如LC - MS的MALDI - TOF和免疫印迹。

using the gelfree 8100 fractionation system for molecular weight-based fractionation with liquid phase recovery

GELFREE 8100分馏系统是一种新的蛋白质分馏系统的设计，以最大限度地提高蛋白质在分子量基于分馏恢复。该系统由单用，8样本容量墨盒和台式GELFREE分馏仪器。在分离，应用的阳极和阴极水库之间的电压恒定，每个蛋白质混合物到一个特别设计的凝胶柱凝胶电泳从装载室的驱动。蛋白质都集中到一个严密乐队在浓缩胶和分离的基础上各自的一个解决凝胶电泳迁移率。蛋白质洗脱列，他们被困在液相集中在收集室， 免费的凝胶。该仪器是在特定的时间间隔，然后暂停，并用吸管收集分数。重复这一过程，直到所有需要的分数已收集。如果少于8个样本的墨盒上运行，可用于任何未使用的商会，在随后的分离。 这种新颖的技术，有利于快速和简单的分离复杂的蛋白质混合物的同时，和以前提供的分馏方法相比，提供了多种优势。该系统能够分馏高达1mg的每通道的总蛋白，每个墨盒8mg。较广泛的群众范围内的完整蛋白质的分离分子量的基础上，保留重要的分析物的理化性质。液相包封提供，同时消除了乐队或现场切割的需要，使分馏过程的高度重复性1，回收率高。

Watch this Scientific Journal Video about Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery at JoVE.com

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

影随行介绍GELFREE 8100分馏系统的使用，哪些分区复杂的蛋白质样品分子量的基础上，并恢复在液相组分。视频描述技术如何工作，如何使用它，并提供与分割牛的肝组织匀浆的聚丙烯酰胺凝胶电泳分析得到的数据，。

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

using-gelfree-8100-fractionation-system-for-molecular-weight-based

Research

JoVE Journal

Biology

16.4K 次观看.  Protein Discovery, Inc.. 影随行介绍GELFREE 8100分馏系统的使用，哪些分区复杂的蛋白质样品分子量的基础上，并恢复在液相组分。视频描述技术如何工作，如何使用它，并提供与分割牛的肝组织匀浆的聚丙烯酰胺凝胶电泳分析得到的数据，。

视频: Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

制备及分馏爪蟾卵提取

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

科研

生物学

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

可视化线虫角质层结构的亲脂性的重要染料，直接投资收益

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

精子冷冻保存和恢复使用I ·冷冻包

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

液相亲和捕获试验用磁珠研究蛋白质相互作用:脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

研究蛋白质的质量控制体系<em&gt;Ð。菌</em&gt;使用温控活细胞成像

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the mitochondrial genome. MDPs have a wide variety of biological effects such as protecting neurons from apoptosis, improving metabolic markers, and protecting cells from chemotherapy. Humanin was the first MDP to be discovered and is the most studied peptide among the MDP family. The membrane receptors and downstream signaling pathways of humanin have been carefully characterized. Additional MDPs such as MOTS-c and SHLP1-6 have been more recently discovered and the signaling mechanisms have yet to be elucidated. Here we describe a cell culture based method to determine the function of these peptides. In particular, cell fractionation techniques in combination with western blotting allow for the quantitative determination of activation and translocation of important signaling molecule. While there are other methods of cell fractionation, the one described here is an easy and straightforward method. These methods can be used to further elucidate the mechanism of action of these peptides and other therapeutic agents.

This protocol describes how to stimulate cells with mitochondrial-derived peptides and assess the signaling cascade and localization of phospho-proteins.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

摘要

探索更多视频

此视频中的章节