This work was supported by a National Institutes of Health grant (GM77311) and a seed grant from The Ohio Cancer Research Associates (to H.A.F.). SM was partially supported by an Up on the Roof fellowship from the Human Cancer Genetics Program of The Ohio State University Comprehensive Cancer Center.

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The authors declare that they have no competing financial interests.

المجهر الكمي في بيولوجيا الخلية ويرتبط عادة مع فحوصات التصوير الخلية الحية، مثل نقل الإسفار الرنين الطاقة (الحنق)، الإسفار الانتعاش بعد photobleaching من (FRAP)، وما إلى ذلك، هناك أمثلة متزايد من علماء الأحياء الخلايا نامية مختلفة المقايسات المجهري الكمية لثابت الخلايا...

تتكون Centrosomes من زوج من centrioles محاطة المواد محيط بالمريكز (PCM). يجري المراكز الرئيسية أنيبيب المنظمة (MTOCs) في خلايا الثدييات، centrosomes بمثابة قطبي مغزل الإنقسامية في تقسيم الخلايا، وبالتالي تساعد على الحفاظ على سلامة الجينوم 1. في الخلايا هادئة (على سبيل المثال، خلال مرحلة G0)، واحد من اثنين من centrioles من الجسيم المركزي، وهي مريكز الأم، وتحويلها إلى هيئة القاعدية لتجميع هدب الابتدائي، عضية الحسية جاحظ الخروج من سطح الخلية 2. مرة واحدة الخلايا إعادة إدخال دورة الخلية، ويتم تفكيكها أهداب الابتدائي وكل مريكز يوجه التجمع من طليعة المريكز في نهايته القريبة أن يستطيل تدريجيا لتشكيل مريكز ناضجة 3. في بداية S-المرحلة، يتم تشكيل بنية تشبه عجلة العربة التي توفر التماثل 9 أضعاف إلى مريكز على سطح كل مريكز القائمة وسوف تصبح قاعدة كل طليعة المريكز. Sas6 رقبعة أمر لا غنى عنه ليتم تجنيده التجمع مريكز لتشكيل محور عجلة العربة 4-6. ثم يتم تجميع البروتينات مريكزي أخرى على عجلة العربة في درجة عالية من التنظيم، الأقرب إلى الطريقة البعيدة 7. بعد الانتهاء بالضبط الازدواجية مريكز، وخلايا تجميع المواد محيط بالمريكز إضافية لبناء اثنين centrosomes وظيفية بحلول نهاية المرحلة G2 8. وبالإضافة إلى المكونات الأساسية مريكزي 9-11، والعديد من البروتينات الأخرى بما في ذلك تحركات، الفسفاتازات، مرافقين، مكونات سقالة، غشاء البروتينات المرتبطة بها والآلات تدهور ترتبط مع centrioles والهيئات القاعدية وPCM في أوقات مختلفة من دورة الخلية 12-16. ويلاحظ في كثير من الأحيان أن مستويات centrosomal من العديد من البروتينات تنظم زمنيا من قبل آليات الاستهداف centrosomal و / أو تدهور proteasomal في centrosomes. الأهم من ذلك أن التقلبات في مستوى centrosomal من عدة بروتينات مثل Plk4، Mps1، Sas6، وCP110 لنقاط ر مختلفة من دورة الخلية ويبدو أن تكون حاسمة لتنظيم التجمع مريكز 5،17-22، وفي حالة Mps1 منع هذا التدهور centrosomal يؤدي إلى تشكيل centrioles الزائدة 19. من ناحية أخرى، فإن الكسور centrosomal من عدة بروتينات أقل عطوب بالمقارنة مع حمامات عصاري خلوي. على سبيل المثال سيرنا بوساطة أسفل تنظيم Centrin 2 (Cetn2) أدى إلى سوى انخفاض معتدل من مستوى البروتين في centrioles على الرغم من انخفاض كبير في مستوياته خلية كاملة 23. ولذا فمن الأهمية بمكان لقياس التغيرات في مستوى البروتينات centrosomal في الجسيم المركزي بدلا من قياس مستويات البروتين خلية كاملة عند تقييم وظائف الجسيم المركزي الخاصة بهم. في هذه الدراسة قمنا بتطوير فحص باستخدام المناعي غير المباشر (معهد التمويل الدولي) لتحديد المستوى النسبي للبروتين في centrosomes. تم تطوير هذا الفحص بشكل خاص لتحليل الخلايا التي هي من عينات مختلفةوبالتالي لا يمكن تصويرها في نفس الوقت. ويمكن لهذه العينات أن تكون الخلايا التي تم التعامل مع الكواشف المختلفة (أي المخدرات مقابل السيطرة)، التي تم جمعها في نقاط زمنية مختلفة (أي نبض مقابل مطاردة)، أو هي في مراحل مختلفة من دورة الخلية. مبدأ هذا الاختبار يكمن في قياس الخلفية تصحيح كثافة الفلورسنت المقابلة لبروتين في منطقة صغيرة وتطبيع تلك القيمة ضد نفسه للبروتين آخر والتي لا تختلف في ظل الظروف التجريبية اختيار المستويات. وقد استخدمت العديد من الدراسات في علم الأحياء الجسيم المركزي مؤخرا مختلف تقنيات المجهر الكمية، في كل الخلايا الحية أو الثابتة، لتحديد وظيفة الجسيم المركزي محددة من البروتينات مرشح 24-27. مماثلة لتلك المقايسات، والتقنية الحالية أيضا يقيس الخلفية تصحيح كثافة مضان من البروتين الاختبار. ومع ذلك، فإن إدراج تطبيع باستخدام معيار الداخلي في هذا الاختبار من المرجح تقديم أكبردقة وثقة في تحليل عينتين المختلفة التي هي على اثنين من coverslips مختلفة. وعلاوة على ذلك، بالإضافة إلى فحص مستوى البروتين في centrosomes، مع تعديلات طفيفة هذه الطريقة يمكن تطبيقها على مجموعة متنوعة من الظروف التجريبية أو في مواقع الخلوية الأخرى. هنا، ونحن لدينا الجمع بين الكمي المجهري الفحص مع استراتيجية نبض مطاردة BrdU لمقارنة الخلايا من مختلف مراحل دورة الخلية. بدلا من استخدام التقنيات القياسية اعتقال دورة الخلية وإطلاق لدراسة مختلف نقاط زمنية دورة الخلية، وخلايا المتزايد بشكل غير متزامن يتم تحضين مع BrdU لتسمية الخلايا في S-المرحلة، ومطاردة الخلايا المسمى لأوقات مختلفة (عادة 4-6 ساعة). ومعظم الخلايا المسمى يكون في S-المرحلة مباشرة بعد النبض. يتم اختيار طول مطاردة وذلك بعد مطاردة، الخلايا المسمى سيكون في أواخر S، G2، أو الانقسام، والتي يمكن التحقق منها من قبل الخصائص المورفولوجية مثل هذا الموقف الفلك من centrosomes فيما يتعلق مجلس التوحيد الوطنىليو، وبعد المسافة بين centrosomes، والتكثيف من الكروموسومات الخ وهكذا، وطول مطاردة يعتمد على متوسط ​​مدة S، M G2 ومرحلة من نوع خلية معينة. منذ هذا النهج يتجنب مثبطات دورة الخلية مثل هيدروكسي يوريا، aphidicolin، نوكودازول، وما إلى ذلك، يتيح تحليل دورة الخلية أكثر أهمية من الناحية الفسيولوجية. وبالتالي، علينا أن نظهر هنا أن الكمية الفحص المجهري مضان وحدها، أو بالاشتراك مع BrdU نبض مطاردة الفحص، هي تقنية بسيطة لكنها قوية لقياس بدقة التغيرات النسبية في مستوى centrosomal من بروتين مرشح خلال دورة الخلية رابط الجأش. قمنا بقياس مستوى centrosomal من VDAC3، وهو البروتين الذي حددنا مؤخرا في centrosomes بالإضافة إلى الميتوكوندريا 16،28، وذلك باستخدام هذه المقايسات. النتائج التي تم الحصول عليها هنا تحقق من ملاحظتنا السابقة أن تجمع centrosomal من VDAC3 ينظمه تدهور، وتختلف أيضا في dependen دورة الخليةر بطريقة 16 والتأكد من صحتها وعلاوة على ذلك إمكانية تطبيق هذا الأسلوب.

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quantitative immunofluorescence assay to measure the variation in protein levels at centrosomes

Centrosomes هي عضيات صغيرة ولكنها مهمة التي تشكل أقطاب الإنقسامية المغزل للحفاظ على سلامة الجينوم أو التجمع أهداب الأساسي لتسهيل مهام الحسية في الخلايا. يجوز تنظيم مستوى بروتين مختلف في centrosomes من في مواقع .cellular أخرى، والاختلاف في مستوى centrosomal من عدة بروتينات في نقاط مختلفة من دورة الخلية ويبدو أن تكون حاسمة لتنظيم السليم التجمع مريكز. قمنا بتطوير مضان الكمي المجهري الفحص الذي يقيس التغيرات النسبية في مستوى البروتين في centrosomes في الخلايا الثابتة من عينات مختلفة، مثل في مراحل مختلفة من دورة الخلية أو بعد العلاج مع مختلف الكواشف. مبدأ هذا الاختبار يكمن في قياس الخلفية تصحيح كثافة الفلورسنت المقابلة لبروتين في منطقة صغيرة، وتطبيع أن قياس ضد نفسه للبروتين آخر لا تختلف تحت ج التجريبية المختارةondition. استخدام هذا الاختبار في تركيبة مع نبض BrdU واستراتيجية مطاردة لدراسة دورات خلية رابط الجأش، لدينا التحقق من صحة كميا الملاحظة الأخيرة في أن ينظم التجمع centrosomal من VDAC3 في centrosomes خلال دورة الخلية، من المحتمل بسبب تدهور بوساطة proteasome وتحديدا في centrosomes.

حددت لدينا دراسات حديثة توطين centrosomal الرواية وظيفة VDAC3، واحدة من porins الميتوكوندريا 16،28. أظهر المناعية من عدة خلايا الثدييات بما في ذلك الخلايا RPE1 باستخدام الأجسام المضادة VDAC3 محددة تلطيخ centrosomal بارز وضعيفة نسبيا تلطيخ الميتوكوندريا. أظهرنا أيضا أن VDAC3 centrosomal يرت...

Watch this Scientific Journal Video about Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes at JoVE.com

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

الكمية المناعي الفحص لقياس التفاوت في مستويات البروتين في Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

quantitative-immunofluorescence-assay-to-measure-variation-protein

Research

JoVE Journal

Biology

14.9K Views.  The Ohio State University. Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein’s fluorescence intensity to that of an appropriate internal standard. 

Video: Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Photobleaching فحوصات (FRAP و FLIP) لقياس حيوية الكروماتين البروتين في الخلايا الحية الجذعية الجنينية

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

على البروتين MultiBac مجمع منصة الإنتاج في EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

تدفق تحليل Cytometric من التكملة الإسفار ثنائي الجزيء: عالية الإنتاجية الأسلوب الكمي لدراسة التفاعل البروتين البروتين

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

Centrosomes التصوير في يطير الخصية

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

قياس مستويات هيم في تخليق خلايا الثدييات

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

في المختبر مثلأيشن الفحص لدراسة البروتين أرجينين مثلأيشن

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

الأخضر الفلورسنت القائم على البروتين التعبير فحص غشاء البروتينات في<em&gt; الإشريكية القولونية</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

في المختبر الفحص لقياس فسفاتيديل إيثانولامين ناقلة ميثيل آخر

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

استخدام أجهزة ميكروفلويديك لقياس عمر والخلوية الظواهر في خلايا الخميرة في مهدها واحدة

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

تقنيات الفحص المجهري [كنفوكل] لدراسة تفاعلات البروتين البروتين وحركية في الحمض النووي آفات متقدمة

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...