우리는 원고의 중요한 읽기 로리 리 감사합니다. TWC는 미국 심장 협회 (American Heart Association) Predoctoral 원정대 (12PRE6590007)에 의해 지원된다. MRB는 국립 암 연구소 교육 교부금 (T32의 CA119925)에 의해 지원된다. SSH는 건강의 국립 연구소 (R01DK078640)에 의해 지원된다. EL는 건강의 국립 연구소 (R01GM081635 및 R01GM103926)에 의해 지원된다.

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저자는 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

Xenopus의 계란 추출물은 β-catenin의 회전율을 조사하기위한 강력한 생화학 적 시스템입니다. Xenopus의 계란 추출물의 β-catenin의 농도는 ~ 25 nm의 2입니다. 최적의 조건 하에서, 계란 추출물은 50 ~ 100 ㎚ / hr의 속도로 β-카테닌을 저하 할 수 있고 200에서 24 nM의 최대량의 절반이다. Xenopus의 계란 추출물을 사용하여 β-catenin의 분해를 성공적으로 재구성에 대한 몇 가지...

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

는 Xenopus laevis의 계란 추출물은 세포 골격 역학, 핵 조립 및 수입, 세포 사멸, 유비퀴틴 대사, 세포주기의 진행, 신호 전달, 단백질 회전율 1-17를 포함하여 많은 세포 생물 학적 과정을 연구하기 위해 광범위하게 사용되어왔다. 계란 추출물은 본질적으로 이러한 프로세스를 실행하고 조사를 활성화하는 데 필요한 모든 필수 세포질 구성 요소가 포함되어 희석 세포질을 나타 내기 때문에 Xenopus의 계란 추출물 시스템은 세포 프로세스의 군단의 생화학 적 분석에 대한 의무입니다. 계란 추출물 많은 양의 물질 ​​(예, 단백질 정제 또는 고 처리량 스크리닝) 18-20의 많은 양을 필요로 생화학 적 조작에 대해 한 번에 제조 할 수있다. 또 다른 장점은 Xenopus의 계란 추출물에서 특정 단백질의 농도가 정확하게 재조합 단백질 및 / 또는 endog immunodepletion의 첨가에 의해 조절 될 수 있다는 것이다또한 단백질의 발현을 제어하기 어렵다 플라스미드 DNA의 형질 대조적 enous 단백질. 또, 사용 가능한 재조합 단백질의 결여는 외생 첨가 mRNA의 번역을 위해 새로 제조 Xenopus의 계란 추출물의 고용량을 활용 관심 단백질을 코딩하는 전 사체의 첨가에 의해 극복 될 수있다. 단백질 분해의 규제는 많은 세포 경로의 조절에 중요합니다 21. Xenopus의 계란 추출물이 시스템은 전사와 번역의 혼란의 영향없이 단백질 회전율을 모니터링 할 수있는 여러 가지 방법에 대해 허용하는 단백질 분해를 연구하기 위해 광범위하게 사용되어왔다을 처리합니다. Wnt 신호 전달 경로 개발과 질병에 중요한 역할을 담당하고 매우 보존 신호 통로입니다. β-카테닌,이 Wnt 경로의 주요한 효과기의 회전율은 매우 증가 된 정상 상태 L에게 규제되고,β-catenin이의 거시기는이 Wnt 표적 유전자의 활성화를 위해 중요합니다. β-catenin의 분해의 중요성은 β-catenin의 분해를 억제로 Wnt 경로에 돌연변이가 ~ 대장 암 (22)의 모든 산발적 인 케이스의 90 %에서 발견되는 사실에 의해 강조 표시됩니다. 이 Wnt 경로의 구성 요소에 의한 β-catenin의 저하가 충실히 매출의 메커니즘을 연구 할뿐만 아니라 분해 2, 19, 20, 23 ~ 29의 새로운 작은 분자 변조기를 식별하는 Xenopus의 계란 추출물에 효과적으로 요약 할 수있다. 세포주기를 공부 Xenopus의 계란 추출물의 제조 방법은 이전 조브 공보 30-32에 기술되었다. 현재 프로토콜은 이러한 방법의 변형을 설명하고의 분해에 최적화되어 있습니다 [35 S] - 방사성 표지 된 β-catenin의와 루시 페라 제 - 태그 β-catenin이있는Xenopus의 계란 추출물. 방사성 동위 원소 분해 분석은 오토 라디오를 통해 단백질 수준을 직접 시각화 할 수 있습니다. [35 S] 메티오닌이어서 직접 분해 반응에 첨가 될 수있는 시험 관내 번역 반응을 사용하여 관심의 단백질에 도입된다. 또한, 방사성 표지 단백질 회전율 분석은 단백질 안정성에 영향을 미칠 수있는 그 단백질 또는 에피토프 태그에 대한 항체를 요구하지 않는다. 단백질 수준에서도 작은 변화, 방사성 표지 된 단백질 밴드의 강도의 변화에 반영 용이 오토 라디오 그래피에 의해 시각되므로 [35 S]를-방사성 표지 분해 분석은 단백질 회전율이 시각화를위한 매우 유용한 방법을 나타낸다. 순서 반딧불 루시페라아제에 β-카테닌의 퓨전 (이하, 간단히 「루시퍼 &quot;라 함), 단백질 수준의 정밀하고 손쉬운 정량적 측정을 허용β-catenin의 회전율 19, 20의 운동 특성을 결정합니다. 루시퍼 라제 분석의 주요 장점은 쉽게 확장되어 강한 양적 시스템을 제공한다는 것이다. 다음 프로토콜은 β-catenin의 분해 및 신규 β-catenin의 변조기의 높은 처리량 검사를위한 강력하고 효율적이며 효과적인 방법을 시금위한 간단한 방법을 제공합니다.

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

는 Xenopus laevis의 계란 추출물. Xenopus의 계란 추출물이 세포주기 및 신호 전달 경로 1-3 포함하여 많은 세포 맥락에서 단백질 회전율을 연구하는 데 사용 된 다양한 세포 과정의 생화학을 공부 잘 특징으로, 강력한 시스템입니다. 여기서, 방법이 중요한로 Wnt 경로의 구성 요소, β-catenin의의 분해를 촉진하기 위해 최적화 된 Xenopus의 계란 추출물을 분리에 설명되어 있습니다. 두 가지 방법은 Xenopus의 계란 추출물에서 β-카테닌 단백질 분해를 평가하기 위해 설명되어 있습니다. 다른 하나는보다 쉽게 높은 처리량 분석 (반딧불 루시 페라 제 - 태그 융합 단백질)에 대해 조정하는 동안 한 가지 방법은, ([35 S] - 방사성 표지 단백질) 시각 정보를합니다. 설명 된 기술은 β-카테닌 단백질 회전율을 평가하고 해당 매출에 공헌 분자 구성 요소를 식별하는 데 사용될 수 있지만, 이에 한정되지 않는다. 또한 ABIL루시 페라 제 - 태그 단백질의 양적 및 손쉬운 판독과 함께 균일 한 Xenopus의 계란 추출물의 큰 볼륨을 정화하는 ITY이 시스템은 쉽게 β-catenin의 분해 변조기에 대한 높은 처리량 검사에 적용 할 수 있습니다.

Xenopus의 계란 추출물의 β-catenin의 저하의 회로도는 그림 2A에 표시됩니다. 35 S 표지 β-catenin의이 Xenopus의 계란 추출물에 배양, 분취 량 (1 ML 상당 추출) 적절한 시간에 제거하고 샘플을 실시 하였다 SDS-PAGE는 오토 라디오 그래피 하였다. 이 Wnt 경로의 구성 요소에 의한 β-catenin의 분해는 유비퀴틴 - 프로 테아 좀 시스템 (2)에 의해 매개되며, Xenopus의 추출?...

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

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

방법은 Xenopus의 계란 추출물 및 단백질 분해의 저분자 변조기에 대한 고 처리량 스크리닝을 위해 그 적응 방사성 표지와 루시퍼 라제 융합 단백질을 이용하여 단백질 분해 분석에 대해 설명한다.

β-catenin의 저하의 재구성<em&gt; Xenopus의</em&gt; 계란 추출

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 조회수.  Vanderbilt University Medical Center.  방법은 Xenopus의 계란 추출물 및 단백질 분해의 저분자 변조기에 대한 고 처리량 스크리닝을 위해 그 적응 방사성 표지와 루시퍼 라제 융합 단백질을 이용하여 단백질 분해 분석에 대해 설명한다. 

비디오: Reconstitution Of &#946;-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.

Xenopus laevis 에그의 작성과 분류는 압축을 풉니다

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

연구

생물학

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.

Fractionated Xenopus 에그 추출물을 사용하여 체외 핵 어셈블리

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 ...

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.

이온 채널 유학을위한 패치 클램프 및 재관류 기법으로 표현 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 ...

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.

사용하여 DNA의 손상 세키의 연구<em&gt; Xenopus</em&gt; 달걀 압축을 풉니 다

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.

생산<em&gt; Xenopus의 tropicalis</em&gt; 달걀은 미세 소관 관련 RNA를 식별하기 위해 추출

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.

의 얼굴 이식<em&gt;는 Xenopus laevis의</em&gt; 태아

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.

<em&gt; Xenopus의</em형광 측정으로&gt; 난자 절개 바셀린 갭 전압 클램프 기술

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

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.

구강 안면 표현형의 정량화에<em&gt; Xenopus의</em

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

정제 된 야생형과 돌연변이 체 CFTR 단백질의 기능의 재구성 및 채널 활동 측정

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).