저자는 자신의 중요한 검토하고, 원고들의 공헌에 대한 샤론 그룹 회원 주셔서 감사합니다. 우리는 Morasha과 Bikura 프로그램, 이스라엘 과학 재단 (부여 Nos. 1823년에서 1807년까지과 378/08), Biomembrane 연구 조셉 콘 미네르바 센터, 뉴 과학자 Chais 가족 펠로 프로그램, 아브라함의 지원 감사 그리고 소니아 Rochlin 재단, 울프슨 가족 자선 신탁, 헬렌과 Biomolecular 구조 및 조립에 밀튼 A. Kimmelman 센터, Shlomo와 사빈 Beirzwinsky의 부동산, Meil​​ 드 Botton 앤슬리, 그리고 카렌 심, 영국.

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+macromolecular+protein+rings+in+the+absence+of+bulk+water.">Evidence for macromolecular protein rings in the absence of bulk water.</a> Science. 310 (5754), 1658-1661 (2005).</li><li>Ruotolo, B. T., Robinson, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspects+of+native+proteins+are+retained+in+vacuum.">Aspects of native proteins are retained in vacuum.</a> Curr Opin Chem Biol. 10 (5), 402-408 (2006).</li><li>Giles, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+a+travelling+wave-based+radio-frequency-only+stacked+ring+ion+guide.">Applications of a travelling wave-based radio-frequency-only stacked ring ion guide.</a> Rapid Commun Mass Spectrom. 18 (20), 2401-2414 (2004).</li><li>McKay, A. R., Ruotolo, B. T., Ilag, L. L., Robinson, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+measurements+of+increased+accuracy+resolve+heterogeneous+populations+of+intact+ribosomes.">Mass measurements of increased accuracy resolve heterogeneous populations of intact ribosomes.</a> J Am Chem Soc. 128 (35), 11433-11442 (2006).</li><li>Ruotolo, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+mobility-mass+spectrometry+reveals+long-lived,+unfolded+intermediates+in+the+dissociation+of+protein+complexes.">Ion mobility-mass spectrometry reveals long-lived, unfolded intermediates in the dissociation of protein complexes.</a> Angew Chem Int Ed Engl. 46 (42), 8001-8004 (2007).</li><li>Morton, V. L., Stockley, P. G., Stonehouse, N. J., Ashcroft, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+virus+capsid+assembly+from+non-covalent+mass+spectrometry.">Insights into virus capsid assembly from non-covalent mass spectrometry.</a> Mass Spectrom Rev. 27 (6), 575-595 (2008).</li><li>Valentine, S. J., Counterman, A. E., Clemmer, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+database+of+660+peptide+ion+cross+sections:+use+of+intrinsic+size+parameters+for+bona+fide+predictions+of+cross+sections.">A database of 660 peptide ion cross sections: use of intrinsic size parameters for bona fide predictions of cross sections.</a> J Am Soc Mass Spectrom. 10 (11), 1188-1211 (1999).</li><li>Mesleh, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+information+from+ion+mobility+measurements:+effects+of+the+long-range+potential.">Structural information from ion mobility measurements: effects of the long-range potential.</a> J Phys Chem. 100, 16082-16086 (1996).</li><li>Shvartsburg, A. A., Jarrold, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+exact+hard-spheres+scattering+model+for+the+mobilities+of+polyatomic+ions.">An exact hard-spheres scattering model for the mobilities of polyatomic ions.</a> Chem Phys Lett. 261, 86-91 (1996).</li></ol>

관심 없음 충돌 선언하지 않습니다.

프로토콜 여기서 설명하는 것은 자신의 전반적인 모양, subunit 포장 및 토폴로지에 대한 정보를 제공하는 목적으로, 알 수없는 입체 구조 단백질 또는 단백질 단지의 충돌 크로스 섹션을 정의할 수 있습니다. 이를 위해 충돌 단면적 값이 일단은 구조 세부 사항이 값을 변환하는 데 필요한 그려져 있습니다. 이 과정은 추가적인 실험의 노력뿐만 아니라 간략하게 아래에 설명하는 전산 분석을 필요로합니다. 로 시작하기 위해, 그것은 알려진 구조 단백질 또는 단백질 단지를 분석하는 것이 좋습니다. 이러한 측정 방법의 유용 품질 관리를 제공할 수 있으며, 이론적 측정 Ω 값을 비교하여 인수 매개 변수의 정확성 평가를 활성화합니다. 이론 교차 단면 영역은 결정 구조에서 계산 수있는 연산자는 필요에 따라 코드 수정을 허용 오픈 소스 FORTRAN 기반 소프트웨어입니다 MOBCAL 15,16 소프트웨어를 사용하여 조정합니다. 이러한 계산을 실행하는 그것은 입력 구조마다 반복 수행 계산의 숫자가 증가하고 원자의 다수를 포함하는 조정 파일이 하나를 수락하는 것을 이러한 프로그램을 수정해야합니다. 복수 어셈블리 내에서 subunits의 topological 준비를 정의하는 IM - MS의 전략은 최근 4,6을 제안되었습니다. 방법은 더 작은 구성 요소를 단백질 분리 어셈블리 경로의 모니터링을 포함한다. 이 분리는 어셈블리의 &quot;빌딩 블록&quot;의 반사 subcomplexes의 배포에 상승을 제공 솔루션 위상 조건을 통제 조정을 통해 이루어진다. 손상 복잡하고 분해 제품의 Ω 값의 동시 측정 후 단백질 단지의 topological 모델을 계산에 사용되는 구조적 지지대를 생성합니다. 이 방법론을 기본의 기본 가정은 생성 subcomplexes들이 네이티브와 같은 주문 확인을 유지하고, 실제로 최근 연구 해체 제품의 솔루션 구조가 유지되는 증명하고 어느 솔루션 또는 가스 단계에 주요 다시 정리하기는 4,6 발생하지 않았는 점이다. 가스 상 단백질 복합 이온에 제사기 구조의 과제의 마지막 단계는 컴퓨터 생성 모델에 충돌 단면적 값을 피팅입니다. 모델링 접근 방식은 다른 가능한 topological subunits의 준비 및 silico Ω의 값을 계산하고 실험 것들에 비교를 탐험하기 위해 고용하고 있습니다. 현재 몇 전산 방법은 subunits 1,8의 직경을 대략 spheretype 결이 거친 방법처럼 사용됩니다. 전체에서이 필드는 초기에 아직 추가적인 개발이 접근법은 일반적인, 그리고 단지의 광범위한 적용하기 위해 필요합니다.

t-wave ion mobility-mass spectrometry: basic experimental procedures for protein complex analysis

이온 이동성 (IM)는 약한 전기장의 영향 하에서 압력 셀을 통과하는 이온을 위해 촬영 시간을 측정하는 방법입니다. 이온이 표류 영역을 통과하는 속도는 자신의 크기에 따라 달라집니다 큰 이온은 작은을 구성하는 이들 이온보다 배경 불활성 가스 (일반적으로 N 2)와 충돌이 더 많은 경험을하기 때문에 메신저 장치를 통해 더 천천히 여행을합니다 단면. 일반적으로 시간이 그것은 그들의 충돌 단면 (Ω)에 따르면, 치밀한 가스상 그들을 구분하지만 마이 그 레이션하는 이온에 대한 소요됩니다. 최근 IM 분광법은 질량 분광법과이 여행 - 웨이브 (T - 파) Synapt 이온 이동성 질량 분석계 (IM - MS)가 출시되었습니다와 결합했다. 이온의 이동성과 질량 분광법을 통합하는 것은 3 차원 스펙트럼을 (유료, 강도, 그리고 표류 시간 질량) 항복, 샘플 분리와 정의의 여분 차원을 수 있습니다. 이 분리 기술은 스펙트럼 중복 감소 수 있도록, 매우 유사한 질량, 또는 대량으로 충전 비율지만, 다른 드리프트 시간으로 이기종 단지의 해상도를 수 있습니다. Ω이 이온의 전반적인 모양과 토폴로지에 관련되어 같이 또한, 드리프트 시간 측정, 구조 정보의 중요한 계층을 제공합니다. 측정된 드리프트 시간 가치와 Ω 사이의 상관 관계가 정의 교차 섹션 1 calibrant 단백질에서 생성된 교정 곡선을 사용하여 계산됩니다. IM - MS 방식의 전원이 subunit의 포장 및 micromolar 농도에서 단백질 어셈블리의 전체 모양을 정의하는 능력에 달려있다, 그리고 거의 생리적 조건 1. 개별 단백질 2,3 및 비 공유 결합 단백질 단지 4-9 모두 몇 가지 최근의 메신저 연구는 성공적으로 단백질 제사기 구조는 가스 단계에서 유지되는 시연, 그리고 알 수없는 형상의 단백질 어셈블리의 연구에서이 방법의 가능성을 강조 . 10, 여기, 우리는 Synapt를 사용하여 단백질 단지의 IMS - MS 분석 (Quadrupole 이온 이동성 - 시간의 기내) HDMS 악기 (유일한 상용 IM - MS 장비 현재 워터스 주)에 대한 자세한 설명을 제공합니다. 우리는 기본적인 최적화 단계, 충돌 크로스 섹션의 교정, 데이터 처리 및 해석에 대한 방법을 설명합니다. 프로토콜의 마지막 단계는 이론적 Ω의 값을 계산하는 방법을 설명합니다. 전체 프로토콜 단백질 어셈블리 IM - MS 특성화의 모든 부분을 커버하려고 시도하지 않는다; 오히려, 그 목적은 현장에서 새로운 연구에 대한 방법의 실용적인 측면을 소개하는 것입니다.

Watch this Scientific Journal Video about T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis at JoVE.com

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

이온 이동성 - 질량 분광법는 충돌 단면 및 질량에 따라 이온을 분리 신흥 가스 상 기술입니다. 방법은 단백질 단지의 전체 토폴로지와 모양에 대한 입체적인 정보를 제공합니다. 여기, 우리는 악기 설정 및 최적화, 드리프트 시대의 교정 및 데이터 해석을위한 기본 절차를 설명합니다.

T - 파 이온 모빌리티 분석법 - 질량 : 단백질 복잡한 분석을위한 기본 실험 절차

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

t-wave-ion-mobility-mass-spectrometry-basic-experimental-procedures

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24.3K 조회수.  Weizmann Institute of Science. 이온 이동성 - 질량 분광법는 충돌 단면 및 질량에 따라 이온을 분리 신흥 가스 상 기술입니다. 방법은 단백질 단지의 전체 토폴로지와 모양에 대한 입체적인 정보를 제공합니다. 여기, 우리는 악기 설정 및 최적화, 드리프트 시대의 교정 및 데이터 해석을위한 기본 절차를 설명합니다.

비디오: T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

든 샘플 준비 : 울트라 씬 레이어 방법

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

연구

생물학

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of cells (approximately 25 in C. borealis) which are well characterized. The cells in the STG exhibit a broad range of outputs and are responsible for the motor actions of the stomach. The stomach contains the gastric mill which breaks down food with three internal teeth, and the pylorus which filters the food before it reaches the midgut. The STG produces two rhythmic outputs to control the gastric mill and pylorus known as central pattern generators (CPGs). Each cell in the STG can participate in one or both of these rhythms. These CPGs allow for the study of neuromodulation, homeostasis, cellular and network variability, network development, and network recovery.
The dissection of the stomatogastric nervous system (STNS) from the Jonah crab (Cancer borealis) is done in two parts; the gross and fine dissection. In the gross dissection the entire stomach is dissected from the crab. During the fine dissection the STNS is extracted from the stomach using a dissection microscope and micro-dissection tools (see figure 1). The STNS includes the STG, the oesophageal ganglion (OG), and the commissural ganglia (CoG) as well as the nerves that innervate the stomach muscles. Here, we show how to perform a complete dissection of the STNS in preparation for an electrophysiology experiment where the cells in the STG would be recorded from intracellularly and the peripheral nerves would be used for extracellular recordings. The proper technique for finding the desired nerves is shown as well as our technique of desheathing the ganglion to reveal the somata and neuropil.

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric nervous system (STNS) of the Jonah crab (C. borealis) can be used for electrophysiology, immunohistochemistry, and cell culture studies. The STNS extraction is done in two parts: the gross and fine dissection.

암 보레알리스 Stomatogastric 신경계의 해부

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

랍스터의 뱃속의 총 해부, Homarus Americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

비침 투 VIVO에서 스몰 애니멀 MRI 및 MRS : 기본 실험 절차

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

구조 질량 분광법에 의해 큰 단백질 단지 분석

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

이온 채널의 돌연변이 유발과 기능 분석 Heterologously 포유 동물 세포에 표현

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

성인 Zebrafish의 생물 화학 분석을위한 혈액 수집

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

기본<em&gt; Caenorhabditis elegans</em&gt; 방법 : 동기화 및 전망

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

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.

EMBL에서 MultiBac 단백질 복잡한 생산 플랫폼

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

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

ChroP 접근은 염색질의 궤적 특정 프로테오믹스 풍경을 해부하는 칩 및 질량 분석을 결합

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...