著者は彼らの批判的検討のため、および原稿への彼らの貢献のためにシャロンのグループメンバーに感謝。我々は、イスラエル科学財団（助成番号1823から1807と378/08）、生体膜研究のためのヨーゼフコーンミネルバセンター、新しい科学者のためのChaisファミリーフェロープログラム、アブラハムMorashaとBikuraプログラムの支援に感謝していますとソニアRochlin財団、ウォルフソンのファミリー公益信託、ヘレンと生体分子の構造や組立のためのミルトンA. Kimmelmanセンター、シュロモとサビーヌBeirzwinskyの不動産、MEILデボタンエインズレイ、そしてカレンシェムリアップ、英国。

<ol>
	<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+analysis+of+large+proteincomplexes.">Ion mobility-mass spectrometry analysis of large proteincomplexes.</a> Nat Protoc. 3 (7), 1139-1152 (2008).</li><li>Scarff, C. A., Thalassinos, K., Hilton, G. R., Scrivens, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Travelling+wave+ion+mobility+mass+spectrometry+studies+of+protein+structure:+biological+significance+and+comparison+with+X-ray+crystallography+and+nuclear+magnetic+resonance+spectroscopy+measurements.">Travelling wave ion mobility mass spectrometry studies of protein structure: biological significance and comparison with X-ray crystallography and nuclear magnetic resonance spectroscopy measurements.</a> Rapid Commun Mass Spectrom. 22 (20), 3297-3304 (2008).</li><li>Smith, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+drift+time+measurements+from+travelling+wave+ion+mobility+spectrometry-mass+spectrometry+studies.">Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies.</a> Eur J Mass Spectrom (Chichester, Eng). 15 (2), 113-130 (2009).</li><li>Leary, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+measuring+conformation+of+solvent-disrupted+protein+subunits+using+T-WAVE+ion+mobility+MS:+an+investigation+into+eukaryotic+initiation+factors.">Methodology for measuring conformation of solvent-disrupted protein subunits using T-WAVE ion mobility MS: an investigation into eukaryotic initiation factors.</a> J Am Soc Mass Spectrom. 20 (9), 1699-1706 (2009).</li><li>Lorenzen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+stoichiometry+and+conformational+changes+in+the+first+step+of+the+P22+tail+assembly.">Determination of stoichiometry and conformational changes in the first step of the P22 tail assembly.</a> J Mol Biol. 379 (2), 385-396 (2008).</li><li>Pukala, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit+architecture+of+multiprotein+assemblies+determined+using+restraints+from+gas-phase+measurements.">Subunit architecture of multiprotein assemblies determined using restraints from gas-phase measurements.</a> Structure. 17 (9), 1235-1243 (2009).</li><li>van Duijn, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperonin+complexes+monitored+by+ion+mobility+mass+spectrometry.">Chaperonin complexes monitored by ion mobility mass spectrometry.</a> J Am Chem Soc. 131 (4), 1452-1459 (2009).</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=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>

利害の衝突は宣言されません。

ここで説明するプロトコルは、その全体の形状、ユニットのパッキンとトポロジーに関する情報を提供する目的で、未知の三次元構造を持つタンパク質やタンパク質複合体の衝突断面積を定義することができます。この目的のために衝突断面積の値は一度それが構造的な細部にこれらの値を数値に変換する必要が描かれている。このプロセスは、追加の実験的な取り組みだけでなく、以下に簡単に説明されている計算分析を、必要になります。 で始まるには、公知の構造を持つタンパク質やタンパク質複合体を分析することをお勧めします。これらの測定は、方法論の有用な品質管理を提供することができますし、理論と実測Ωの値を比較することにより、取得パラメータの精度評価を可能にします。理論的な断面積は、結晶構造から計算することができますMOBCAL 15,16ソフトウェアを使用して座標を、オペレータのニーズに応じてコードの編集を可能にするオープンソースFORTRANベースのソフトウェアである。このような計算を実行するためには、入力構造ごとに実行反復計算の数が増えると原子の多数を含む座標ファイルは、1を受け入れていることをされるようにプログラムを変更するために必要です。 多成分のアセンブリ内でサブユニットの位相的な取り決めを定義するためのIM - MSの戦略は、最近4,6に提案されている。メソッドは小さいコンポーネントにタンパク質のアセンブリの解離経路の監視を行います。この解離は、アセンブリの&quot;ビルディングブロック&quot;の反射subcomplexesの分布を生じさせる溶液相の条件の制御による調整、によって達成されます。無傷の複雑かつ分解製品の両方のΩ値の同時測定は、タンパク質複合体の位相モデルを計算するために使用されている構造的な制約を生成します。この方法論の根底にある基本的な前提は、生成されたsubcomplexesが母国のような確認を保持することであり、そして実際に最近の研究では、分解製品の溶液構造が維持されることが実証されていると、溶液または気相のどちらかに大きな転位4,6を発生していません。 気相タンパク質の錯イオンに四次構造の割り当ての最後のステップは、コンピュータが生成モデルに衝突断面積の値は継手です。モデリングのアプローチが異なる可能トポロジーサブユニットの手配、彼らのシリコ Ωの値での計算と実験値と比較さを探求するために採用されています。現在はわずか数計算のアプローチはサブユニット1,8の直径に近似するspheretype粗粒度方式と同様に、使用されています。全体的に、このフィールドは、初期の頃のままであり、さらなる発展は、このアプローチは一般的な、そして複合体の広い範囲に適用可能にするために必要です。

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

イオンモビリティ（IM）は、弱電界の影響下で加圧セルを通過するイオンにかかる時間を測定する方法です。イオンがドリフト領域を横断これにより、速度はその大きさに依存します：大規模なイオンがバックグラウンド不活性ガス（通常はN 2）との衝突の大きい数を経験し、その結果、より小さいを構成するこれらのイオンよりもIMのデバイスを介して、よりゆっくりと旅する断面。一般的に、イオンが濃いガスの相も移行するのにかかる時間は、その衝突断面積（Ω）によると、それらを分離する。 最近、IM分析法は質量分析法と結合され、進行波（T波）SYNAPTイオンモビリティー質量分析計（IM - MS）がリリースされました。イオン移動度と質量分析を統合することで三次元スペクトルを（電荷の質量、強度、およびドリフトの時間）を得た、サンプルの分離と定義の余分な次元が可能になります。この分離技術は、スペクトルの重複が減少することができます、と非常に似て質量、または質量対電荷比が、異なるドリフト時間を持つ異種複合体の分解能を可能にします。また、ドリフト時間の測定値は、Ωがイオンの全体的な形状とトポロジーに関連しているとして、構造情報の重要なレイヤを提供しています。測定されたドリフトの時間値とΩの間に相関関係が定義されているクロスセクション1の較正タンパク質から生成された検量線を用いて計算されます。 IM - MSのアプローチのパワーは、サブユニットのパッキングとマイクロモル濃度でタンパク質のアセンブリの全体的な形状を定義する能力にあります、そして近い生理的な条件は1。成功したタンパク質四次構造を気相で維持、および未知の幾何学のタンパク質集合体の研究ではこのアプローチの可能性をハイライト表示されていることを明らかに個々の蛋白質2,3および非共有結合タンパク質複合体4-9、両方の最近のいくつかのIMの研究。 10;ここで、我々は、SYNAPT（四重極イオンモビリティ-飛行時間型）HDMSの楽器（現在入手可能な唯一の商用IM - MSの楽器ウォーターズ株式会社）を用いて、タンパク質複合体のIMS - MS分析の詳細な説明を提供しています。我々は、基本的な最適化の手順、衝突断面のキャリブレーション、およびデータの処理および解釈するための方法を説明します。プロトコルの最後のステップは、理論上のΩ値を算出する方法を説明します。全体的に、プロトコルは、タンパク質のアセンブリのIM - MSの特性評価のあらゆる側面をカバーしようとするのではなく、その目標は、フィールドでの新たな研究者に法の実用的側面を導入することである。

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

イオンモビリティー質量分析法は、その衝突断面積と質量に基づいて、イオンを分離する新興気相技術です。方法は、タンパク質複合体の全体的なトポロジーと形状の三次元情報を提供します。ここで、我々は、機器設定や最適化、ドリフト時間のキャリブレーション、およびデータ解釈のための基本的な手順を概説する。

T波イオンモビリティー質量分析：タンパク質複合体解析のための基本的な実験手順

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

Research

JoVE Journal

Biology

24.3K ビュー.  Weizmann Institute of Science. イオンモビリティー質量分析法は、その衝突断面積と質量に基づいて、イオンを分離する新興気相技術です。方法は、タンパク質複合体の全体的なトポロジーと形状の三次元情報を提供します。ここで、我々は、機器設定や最適化、ドリフト時間のキャリブレーション、およびデータ解釈のための基本的な手順を概説する。

ビデオ: 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).

MALDIサンプル調製：超薄層法

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

生物学

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.

癌ボレアリス口胃神経系の解剖

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).

ロブスターの胃の肉眼解剖、ロブスター属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.

非侵襲的インビボでの小動物MRIとMRS：基本的な実験手順

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.

構造質量分析法によって大きなタンパク質複合体の分析

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.

異種哺乳動物細胞で発現したイオンチャネルの変異導入と機能解析

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.

大人のゼブラフィッシュの生化学的解析のための採血

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.

基本的な<em&gt;線虫Caenorhabditis elegans</em&gt;方法：同期との観測

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.

EMBLでMultiBacタンパク質複合体生産プラットフォーム

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アプローチは、クロマチンの遺伝子座特異的プロテオームの風景を分析するために、チップと質量分析を組み合わせ

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