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يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة. المؤلف، ويتولد زيمانسكي، هو موظف في معهد ماكس بلانك للفسيولوجيا النبات الجزيئية. المؤلف، فالتراود شولز هو موظف في جامعة هوهنهايم في ألمانيا.

بروتوكول المعروضة في هذه الورقة تحتوي على العديد من الخطوات وجميعهم من الأهمية بمكان الحصول على تجزئة نقية وممثل للغشاء البلازما النباتية في أغشية مقاومة المنظفات والمنظفات كسور قابلة للذوبان. وبالتالي، فمن المهم أن تتبع كل خطوة وفقا للتعليمات. <p class="jove_content" style=";...

في عام 1972، اقترح جوناثان سينغر ونيكلسون نموذج غارث فسيفساء السائل نموذج هيكل الأغشية الخلوية، ليحل محل النموذج ساندويتش البروتين الدهني البروتين الذي تم المقبولة عموما في أوائل 1960s. المغني ونيكلسون افترض أن غشاء بيولوجي يمكن اعتباره السائل ثنائي الأبعاد حيث نشر كل الدهون والبروتينات جزيئات بحرية وبسهولة 5. منذ ذلك الوقت، نموذج هيكل غشاء البلازما والمعرفة لتكوين غشاء أصبح أكثر تعقيدا. بشكل خاص، داخل غشاء البلازما، والهياكل مثل المجمعات البروتين والدهون / ستيرول مقرها microdomains المختلين هيكليا يمكن ملاحظتها. في الأغشية النموذج الصناعي 6،7، يمكن الجامدة والدهون الإسفنجية فصل أفقيا من الأنواع الدهون الأخرى لتشكيل الأقاليم مع ميزات المادية المتغيرة. ويتسبب هذا الفصل أساسا داخل الغشاء الخلوي من خصائص الربط الذاتي بين الجامدة وسا غايةالسلاسل الهيدروكربونية turated من phopsho والدهون الإسفنجية 8. بشكل خاص، يرن ستيرول جامدة تفضل التفاعل مع صلابة والأنواع الدهون المشبعة واستقامة هذه التفاعلات إجبار السلاسل الهيدروكربونية المجاورة إلى أكثر التشكل الموسعة، زيادة سمك الغشاء وصلابة. كان واحدا من السمات يلاحظ عموما من ستيرول التخصيب microdomains غشاء الصلابة سفرهم عند العلاج مع المنظفات غير الأيونية مثل هذا تريتون X-100 أو بريج 35. ويعتقد أن هذه الكسور لتكون متطابقة مع microdomains الغشاء وكانت تسمى أغشية مقاومة المنظفات (DRM) على أساس على طريقة إعداد البيوكيميائية 2. استخدام المنظفات غير أيوني خلال استخراج DRM تلقى بعض الانتقادات مثل إعداد DRM البيوكيميائية قد لا تتوافق مباشرة إلى أي مقصورة غشاء محددة داخل الخلية الحية 9. وخاصة، والمنظفات لنسبة البروتين يبدو حاسما في مثل هذه الاستعدادات، ويمكن ق المنظفات المختلفة، فضلا عن كميات المنظفات المختلفة تسفر عن تكوين مختلفة من المنظفات مقاومة الغشاء جزء 10. ومع ذلك، هناك أدلة على أن الأنواع بروتين معين المنتسبين على وجه التحديد مع هذه المجالات غشاء ستيرول الغنية الخلوية، والتي يتم إثراء هذه البروتينات جيدا في التحضيرات الكيميائية الحيوية المقاومة للكسور الغشاء المنظفات 11. كان جوهر البروتينات التي تم العثور عليها في المصنع DRM كسر، والتي كان وجودها في DRMS ​​ستيرول تعتمد، ولا سيما البروتينات GPI الراسية، مثل البروتينات مثل fasciclin arabinogalactan (إف إل أي إس) وأفراد من عائلة البروتين SKU. كما يشير بعض البروتينات، مثل تحركات مثل مستقبلات أو phospholipases تم العثور على 11. هذه النتائج متسقة مع العديد من الدراسات على البروتين microdomains غشاء الثدييات 12،13. أيضا في النباتات هناك أدلة متزايدة لدور microdomains الغشاء في سياق الاستجابة للضغط النفسي 14 -16. بروتوكول الموصوفة هنا يوفر طريقة قوية للتجزئة من microdomains غشاء البلازما، ويستخدم بشكل خاص على البروتين لتركيز المنظفات التي تسمح لنا لتصوير الاجهاد الناجم التعديلات للستيرول الغنية غشاء مقصورة 4،11،14.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS </td>
		</tr>
		<tr>
			<td>Chemicals were ordered from Sigma-Aldrich unless noted otherwise</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonia (stock 25 % solution)</td>
			<td>WAKO</td>
			<td>010-03166</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TiO2 10 &mu;m</td>
			<td>GL-Science</td>
			<td>5020-75010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Empore Disk C18</td>
			<td>Varian</td>
			<td>12145004</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polyethylene glycol(PEG 3350)</td>
			<td>Sigma</td>
			<td>88276</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dextran T500</td>
			<td>Roth</td>
			<td>9219.2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Promega</td>
			<td>V5113</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail (PIC)</td>
			<td>Sigma</td>
			<td>P9599</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>K15NO3</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>NLM-765-PK</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT </td>
		</tr>
		<tr>
			<td>Optima L-80 XP Ultracentrifuge</td>
			<td>Beckman</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BioTek</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EASY-nLC II nano-Liquid Chromatograph</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LTQ-Orbitrap mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge 5417R</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Speed Vac RVC 2-25</td>
			<td>Christ</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shaker Unimax 2010</td>
			<td>Heidolph</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

metabolic labeling and membrane fractionation for comparative proteomic analysis of arabidopsis thaliana suspension cell cultures

البلازما microdomains غشاء هي الميزات على أساس الخصائص الفيزيائية للدهون وستيرول البيئة ولها أدوار معينة في عمليات الإشارة. استخراج microdomains غشاء التخصيب ستيرول من الخلايا النباتية لتحليل البروتين هو مهمة صعبة ويرجع ذلك أساسا إلى إعداد خطوات متعددة ومصادر التلوث للمن المقصورات الخلوية الأخرى. يشكل غشاء البلازما فقط حوالي 5-20٪ من جميع الأغشية في الخلايا النباتية، وبالتالي عزل عالي النقاء جزء غشاء البلازما يمثل تحديا. ويتضمن الطريقة المستخدمة في كثير من الأحيان مائي التقسيم على مرحلتين في غليكول البولي ايثيلين وديكستران والتي ينتج عنها حويصلات غشاء البلازما بدرجة نقاء 95٪ 1. microdomains غشاء ستيرول الغنية داخل غشاء البلازما هي غير قابلة للذوبان على العلاج مع المنظفات غير أيوني الباردة في درجة الحموضة القلوية. يمكن فصل هذا الكسر غشاء مقاومة للالمنظفات من غشاء البلازما بالجملة من قبل تنبيذ فائق في النحوالتدرج ucrose 2. في وقت لاحق، والبروتينات يمكن استخلاصها من الموجات المنخفضة الكثافة من التدرج السكروز بواسطة الميثانول / كلوروفورم هطول الأمطار. وبعد ذلك يتم هضمها التربسين البروتين المستخرج، المحلاة وتحليلها أخيرا LC-MS/MS. هو الأمثل لدينا بروتوكول استخراج لmicrodomains-ستيرول الغنية لإعداد نظيفة مقاومة للالمنظفات الكسور غشاء الخلية من ثقافات نبات الأرابيدوبسيس thaliana. نستخدم وضع العلامات الأيضية الكامل للنبات الأرابيدوبسيس thaliana الثقافات الخلية التعليق مع K 15 NO 3 كمصدر للنيتروجين فقط للدراسات المقارنة البروتين الكمي التالية المعالجة البيولوجية من الفائدة 3. عن طريق خلط نسب متساوية من الثقافات خلية وصفت وغير المسماة لاستخراج البروتين مشتركة يتم الاحتفاظ تأثير على خطوات إعداد النتيجة النهائية الكمية كحد أدنى. أيضا خسائر ماديه اثناء الاستخراج سوف تؤثر على كل من الرقابة وعينات المعاملة في نفس الطريق، ووبالتالي د نسبة الضوء ويتنفس الببتيد سوف تظل ثابتة. في الطريقة المقترحة إما المسمى أو زراعة الخلايا غير المسماة تخضع لعملية المعالجة البيولوجية، في حين أن الآخر بمثابة السيطرة 4.

مع بروتوكول المعروضة باستخدام المسمى أيضي الثقافات خلية نبات الأرابيدوبسيس فمن الممكن لعزل أغشية البلازما من الأنسجة النباتية (STEP2، أرقام 2 و 4)، وإثراء لمقاومة المنظفات الكسور الغشاء داخل غشاء البلازما (الخطوة 3 أرقام 3 و 5). في وقت لاحق، ويسمح ا...

Watch this Scientific Journal Video about Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures at JoVE.com

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

نحن هنا تصف طريقة قوية للتجزئة أغشية البلازما مصنع المنظفات في الأغشية القابلة للذوبان مقاومة والمنظفات يعتمد على خليط من غير المسماة في الجسم الحي بالكامل و15 N المسمى الثقافات خلية نبات الأرابيدوبسيس thaliana. يتم تطبيق هذا الإجراء للدراسات المقارنة البروتين لفهم العمليات الإشارة.

الأيضية وصفها وغشاء التقطير للالمقارن تحليل البروتين من<em&gt; نبات الأرابيدوبسيس thaliana</em&gt; الثقافات تعليق خلية

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

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

14.0K Views.  Max Plank Institute of Molecular Plant Physiology.  نحن هنا تصف طريقة قوية للتجزئة أغشية البلازما مصنع المنظفات في الأغشية القابلة للذوبان مقاومة والمنظفات يعتمد على خليط من غير المسماة في الجسم الحي بالكامل و15 N المسمى الثقافات خلية نبات الأرابيدوبسيس thaliana. يتم تطبيق هذا ا?...

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

Immunoblot Analysis

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

تحليل طخة مناعية

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the ...

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

دراسة النشوء الأحيائي الأغشية مع الفحص الجيني مراسل Luciferase المستندة

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

التعريفي واستخراج السم من البروتين فيوزاريوم النيابة. الثقافات للدراسات البروتين

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

الأزهار وتراجع تحويل thaliana Arabidopsis لتفحص pTSO2 : β غلوكورونيداز التعبير مراسل جين

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

قياس حركية تقدم دورة الخلية مع وصفها الأيض والتدفق الخلوي

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

تحليل خلية معينة من<em&gt; ل arabidopsis</em&gt; يترك طريق الإسفار خلية المنشط الفرز

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

العزلة الفعالة والسريعة من الأجنة المبكرة مرحلة من<em&gt; thaliana نبات الأرابيدوبسيس</em&gt; بذور

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

الأيضية وصفها من لوسين ريتش كرر تحركات 1 و 2 مع المشعة الفوسفات

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

نهج جديد لتحليل مقارن لMultiprotein مجمعات بناء على<sup&gt; 15</sup&gt; N الأيض وصفها والكمية الطيف الكتلي

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

تلطيخ النسيجية من<em&gt; نبات الأرابيدوبسيس thaliana</em&gt; الخليوي الثانوية ستريت عناصر

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

الأيضية وصفها وغشاء التقطير للالمقارن تحليل البروتين من

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تحليل طخة مناعية

دراسة النشوء الأحيائي الأغشية مع الفحص الجيني مراسل Luciferase المستندة

التعريفي واستخراج السم من البروتين فيوزاريوم النيابة. الثقافات للدراسات البروتين

الأزهار وتراجع تحويل thaliana Arabidopsis لتفحص pTSO2 : β غلوكورونيداز التعبير مراسل جين

قياس حركية تقدم دورة الخلية مع وصفها الأيض والتدفق الخلوي

تحليل خلية معينة من

العزلة الفعالة والسريعة من الأجنة المبكرة مرحلة من

الأيضية وصفها من لوسين ريتش كرر تحركات 1 و 2 مع المشعة الفوسفات

نهج جديد لتحليل مقارن لMultiprotein مجمعات بناء على

تلطيخ النسيجية من