نشكر لوري لي لقراءة نقدية للمخطوطة. معتمد من قبل TWC دكتوراه مسبقا زمالة جمعية القلب الأمريكية (12PRE6590007). ويدعم MRB من منحة تدريب المعهد الوطني للسرطان (T32 CA119925). ويدعم SSH من قبل المعاهد الوطنية للصحة (R01DK078640). ويدعم EL من قبل المعاهد الوطنية للصحة (R01GM081635 وR01GM103926).

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يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة.

القيطم استخراج البيض هو نظام البيوكيميائية قوية للتحقيق في دوران β-catenin. تركيز β-catenin في القيطم استخراج البيض هو ~ 25 نانومتر 2. تحت الظروف المثلى، واستخراج البيض قادر على المهينة β-catenin بمعدل 50-100 نانومتر / ساعة ونصف القصوى، في 200 نانومتر 24. وهناك الع?...

A correction was made to <a href="http://www.jove.com/video/51425">Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract</a>. At the time of publication there were some instances where an incorrect volume notation was used. These instances were corrected from:
2.1.2. Add extract to 1/10 the volume of pelleted antibody or affinity beads (e.g., 20 ml pelleted beads to 200 ml extract). In order to minimize dilution of the extract, withdraw as much liquid from the beads as possible before addition of the extract using gel loading tips with long, tapered tips.
2.2.5. Aliquot the appropriate volumes for degradation assay into pre-chilled microfuge tubes on ice. For radiolabeled &#946;-catenin degradation assays, withdraw 2-5 ml extract for each time point.
3.2.3. At the designated time point, remove 1-5 ml of the sample and mix immediately with SDS sample buffer (5x volume) to stop the reaction. To make sure the degradation reaction is completely terminated, flick tube several times and vortex vigorously.
3.2.4. Perform SDS-PAGE/autoradiography. Run 1 ml equivalents (~50 mg of protein) of the extract for each time point/lane. Degradation of &#946;-catenin in Xenopus egg extract should be evidenced by the time-dependent decrease in intensity of the radiolabeled &#946;-catenin band Figure 2. Quantify results using ImageJ, ImageQuant, or other preferred imaging software if necessary.
4.2.2. Add in vitro-translated &#946;-catenin-luciferase fusion (from 4.1) into prepared Xenopus reaction mix (from 2.2) on ice and mix well as in 3.2.1. NOTE: The activity of the &#946;-catenin luciferase that is added to the extract is typically between 20 - 50,000 relative luminescence units (RLU)/ml of extract (based on measurements obtained from 4.1.2). Starting signal should be approximately 100,000 RLU (2-5 ml of the in vitro-translated &#946;-catenin-luciferase fusion).
to:
2.1.2. Add extract to 1/10 the volume of pelleted antibody or affinity beads (e.g., 20 &#181;l pelleted beads to 200 &#181;l extract). In order to minimize dilution of the extract, withdraw as much liquid from the beads as possible before addition of the extract using gel loading tips with long, tapered tips.
2.2.5. Aliquot the appropriate volumes for degradation assay into pre-chilled microfuge tubes on ice. For radiolabeled &#946;-catenin degradation assays, withdraw 2-5 &#181;l extract for each time point.
3.2.3. At the designated time point, remove 1-5 &#181;l of the sample and mix immediately with SDS sample buffer (5x volume) to stop the reaction. To make sure the degradation reaction is completely terminated, flick tube several times and vortex vigorously.
3.2.4. Perform SDS-PAGE/autoradiography. Run 1 &#181;l equivalents (~50 mg of protein) of the extract for each time point/lane. Degradation of &#946;-catenin in Xenopus egg extract should be evidenced by the time-dependent decrease in intensity of the radiolabeled &#946;-catenin band Figure 2. Quantify results using ImageJ, ImageQuant, or other preferred imaging software if necessary.
4.2.2. Add in vitro-translated &#946;-catenin-luciferase fusion (from 4.1) into prepared Xenopus reaction mix (from 2.2) on ice and mix well as in 3.2.1. NOTE: The activity of the &#946;-catenin luciferase that is added to the extract is typically between 20 - 50,000 relative luminescence units (RLU)/&#181;l of extract (based on measurements obtained from 4.1.2). Starting signal should be approximately 100,000 RLU (2-5 &#181;l of the in vitro-translated &#946;-catenin-luciferase fusion).

Erratum: Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

وقد استخدم القيطم المورق استخراج البيض على نطاق واسع لدراسة العديد من العمليات البيولوجية الخلية بما في ذلك ديناميات هيكل الخلية، والتجمع النووية والاستيراد، وموت الخلايا المبرمج، والتمثيل الغذائي اليوبيكويتين، تقدم دورة الخلية، نقل الإشارة، والبروتين دوران 1-17. والقيطم البيض استخراج النظام قابلة للتحليل كيميائي حيوي من حشد غفير من العمليات الخلوية لاستخراج البيض يمثل أساسا السيتوبلازم غير مخفف يحتوي على كافة مكونات حشوية الأساسية اللازمة لتنفيذ هذه العمليات وتمكين التحقيق. كميات كبيرة من استخراج البيض يمكن أن تكون على استعداد في وقت واحد للتلاعب البيوكيميائية التي تتطلب كميات كبيرة من المواد (على سبيل المثال، وتنقية البروتين أو الفرز الفائق الإنتاجية) 18-20. ميزة أخرى هي أن تركيز بروتينات معينة في القيطم استخراج البيض يمكن تعديلها على وجه التحديد عن طريق إضافة المؤتلف البروتين و / أو immunodepletion من endogالبروتينات enous على النقيض من ترنسفكأيشن من الحمض النووي البلازميد حيث التعبير عن بروتين من الفائدة من الصعب السيطرة عليها. بالإضافة إلى ذلك، عدم وجود البروتينات المؤتلف متوفرة ويمكن التغلب عليها عن طريق إضافة نصوص ترميز البروتين من الفائدة، والاستفادة من قدرة الطازجة القيطم استخراج البيض العالية لترجمة مرنا وأضاف خارجيا. تنظيم تدهور البروتين أمر بالغ الأهمية من أجل السيطرة على العديد من مسارات الخلوية والعمليات 21. وقد استخدم على نطاق واسع القيطم استخراج البيض لدراسة تدهور البروتين كما يسمح النظام عن طرق متعددة لرصد دوران البروتين دون التأثيرات الخلط بين النسخ والترجمة. إشارات مسار WNT هو مسار الإشارات حفظا للغاية أن يلعب أدوارا حاسمة في تطوير والمرض. دوران β-catenin، والمستجيب الرئيسية للمسار WNT، ودرجة عالية من التنظيم، وزادت حالة استقرار لترايفل من β-catenin هو أمر حاسم لتفعيل الجينات المستهدفة WNT. وسلط الضوء على أهمية تدهور β-catenin من حقيقة أن الطفرات في مسار WNT التي تمنع تدهور β-catenin جدت في ~ 90٪ من جميع حالات متفرقة من سرطان القولون والمستقيم 22. تدهور β-catenin بواسطة مكونات من مسار WNT يمكن تلخيصها بإخلاص في استخراج البيض القيطم لدراسة آلية من حجم اعمالها، وكذلك لتحديد جهري جزيء صغير الرواية من تدهورها 2، 19، 20، 23-29. وقد وصفت وسائل لإعداد القيطم استخراج البيض لدراسة دورة الخلية في المنشورات السابقة إن الرب 30-32. يصف البروتوكول الحالي لتعديل هذه الأساليب والأمثل لتدهور [35 S]-β-رديولبلد catenin والموسومة luciferase المراسل β-catenin فيالقيطم استخراج البيض. تدهور فحص رديولبلد يسمح للرؤية مباشرة من مستويات البروتين عن طريق تصوير الإشعاع الذاتي. أدرج [35 S] ميثيونين في البروتين من الفائدة باستخدام ترجمة رد فعل في المختبر التي يمكن بعد ذلك إضافتها مباشرة إلى رد فعل تدهور. بالإضافة إلى ذلك، لا يتطلب البروتين رديولبلد دوران فحص الأجسام المضادة ضد البروتين من الفائدة أو علامة حاتمة، والتي يمكن أن تؤثر على استقرار البروتين. لأنه حتى التغيرات الصغيرة في مستويات البروتين، كما يتجلى في التغيرات في كثافة البروتين رديولبلد الفرقة، وتصور بسهولة عن طريق تصوير الإشعاع الذاتي، و[35 S]-رديولبلد يمثل تدهور فحص وسيلة مفيدة جدا لرؤية بروتين دوران 2. انصهار β-catenin ليراعة luciferase (المشار إليها فيما يلي باسم ببساطة &quot;luciferase المراسل&quot;) يسمح لقياس الكمي الدقيق والسهل من مستويات البروتين، من أجللتحديد الخصائص الحركية للβ-catenin دوران 19، 20. والميزة الرئيسية للمقايسة luciferase المراسل هو أنه يوفر نظام كمي قوية على أن يتم تحجيم بسهولة. يوفر بروتوكول التالية أساليب بسيطة لمعايرة تدهور β-catenin وطريقة قوية وفعالة، وفعالة لالفرز الفائق الإنتاجية من رواية جهري β-catenin.

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reconstitution of &#946;-catenin degradation in xenopus egg extract

القيطم المورق استخراج البيض هو، نظام قوي يتميز جيدا لدراسة الكيمياء الحيوية من العمليات الخلوية المختلفة. وقد استخدمت القيطم استخراج البيض لدراسة دوران البروتين في العديد من السياقات الخلوية، بما في ذلك دورة الخلية ومسارات نقل الإشارة 1-3. هنا، يتم وصف طريقة لعزل القيطم استخراج البيض الذي تم الأمثل لتعزيز تدهور المكون مسار WNT الحرجة، β-catenin. يتم وصف طريقتين مختلفتين لتقدير β-catenin تدهور البروتين في البيض القيطم استخراج. أسلوب واحد هو بالمعلومات بصريا ([35 S]-رديولبلد البروتينات)، في حين أن الآخر يتم تحجيم بسهولة أكبر لفحوصات عالية الإنتاجية (يراعة luciferase المراسل الموسومة البروتينات الانصهار). الأساليب المذكورة يمكن استخدامها ل، ولكن ليس على سبيل الحصر، تقييم β-catenin البروتين دوران وتحديد المكونات الجزيئية التي تسهم في حجم اعمالها. بالإضافة إلى ذلك، أبيإيتي لتنقية كميات كبيرة من متجانسة القيطم استخراج البيض جنبا إلى جنب مع قراءات الكمية والسهل من البروتينات الموسومة luciferase المراسل يسمح هذا النظام ليتم تكييفها بسهولة للكشف عن الإنتاجية العالية للجهري من تدهور β-catenin.

ويرد التخطيطي للتدهور β-catenin في القيطم استخراج البيض في الشكل 2A. وقد حضنت 35 S-β-وصفت catenin في القيطم استخراج البيض، وأزيلت مأخوذة (1 مل ​​استخراج ما يعادلها) في الأوقات المناسبة، وتعرض عينات ل SDS-PAGE تليها تصوير الإشعاع الذاتي. وتوسط تدهور β-catenin ب?...

Watch this Scientific Journal Video about Reconstitution Of Î²-catenin Degradation In Xenopus Egg Extract at JoVE.com

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

ووصف طريقة لتحليل تدهور البروتين باستخدام رديولبلد والبروتينات luciferase المراسل الانصهار في القيطم استخراج البيض والتكيف من أجل الفرز الفائق الإنتاجية للجهري جزيء صغير من تدهور البروتين.

إعادة تشكيل β-catenin تدهور في<em&gt; القيطم</em&gt; مستخلصات البيض

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

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Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Unit 1

Translation: RNA to Protein

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12.0K Views.  Vanderbilt University Medical Center.  ووصف طريقة لتحليل تدهور البروتين باستخدام رديولبلد والبروتينات luciferase المراسل الانصهار في القيطم استخراج البيض والتكيف من أجل الفرز الفائق الإنتاجية للجهري جزيء صغير من تدهور البروتين. 

Video: Reconstitution Of β-catenin Degradation In Xenopus Egg Extract

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.

إعداد والتقطير من البيض مقتطفات القيطم المورق

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

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

في المختبر عن طريق الجمعية النووية مجزأ مقتطفات البيض القيطم

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

تصور التعريب في الحمض النووي الريبي القيطم البويضات

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

أعربت المشبك التصحيح وتقنيات الإرواء لدراسة في قنوات ايون القيطم البويضات

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

دراسة الحمض النووي حاجز الضرر باستخدام<em&gt; القيطم</emمقتطفات البيض&gt;

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

إنتاج<em&gt; القيطم مداري</em&gt; البيض مقتطفات من التعرف على الرنا أنيبيب المرتبطة بها

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

زرع الوجه في<em&gt; القيطم المورق</em&gt; الأجنة

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

ال<em&gt; القيطم</em&gt; سيت قص مفتوحة الفازلين جاب الجهد المشبك تقنية مع التألق

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

في المختبر إعادة تشكيل مجمعات خفيفة حصاد النباتات والطحالب الخضراء

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Quantification of Orofacial Phenotypes in Xenopus

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.

Quantification of Orofacial Phenotypes in Xenopus

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects.

إعادة تشكيل β-catenin تدهور في

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إعداد والتقطير من البيض مقتطفات القيطم المورق

في المختبر عن طريق الجمعية النووية مجزأ مقتطفات البيض القيطم

تصور التعريب في الحمض النووي الريبي القيطم البويضات

أعربت المشبك التصحيح وتقنيات الإرواء لدراسة في قنوات ايون القيطم البويضات

دراسة الحمض النووي حاجز الضرر باستخدام

إنتاج

زرع الوجه في

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في المختبر إعادة تشكيل مجمعات خفيفة حصاد النباتات والطحالب الخضراء

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