Los autores agradecen a los miembros del grupo a Sharon por su revisión crítica, y por sus contribuciones al manuscrito. Estamos muy agradecidos por el apoyo de los Programas de Morasha y bikura, la Israel Science Foundation (Grant N º 1823 / 07 y 378/08), el Josef Cohn Minerva Centro de Investigación biomembrana, la Familia Chais Programa de Becas para los nuevos científicos, el Abraham y Sonia Rochlin Fundación, el Fideicomiso de la Familia Wolfson caridad; el Centro Helen y Milton A. Kimmelman de Estructura Biomolecular y de la Asamblea, la finca de Shlomo y Beirzwinsky Sabine; Meil ​​de Botton Aynsley, y Karen Siem, Reino Unido.

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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. 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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. 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El protocolo aquí descrito permite definir la sección transversal de colisión de las proteínas o complejos de proteínas con un desconocido la estructura tridimensional, con el objetivo de proporcionar información sobre su forma general, el embalaje y la topología de la subunidad. Con este fin, una vez los valores de la sección transversal de colisión se muestran, es necesario convertir estos valores a los elementos estructurales. Este proceso requiere de esfuerzos adicionales experimental, así como el análisis computacional, el cual se describen brevemente a continuación. Para empezar, se recomienda analizar las proteínas o complejos de proteínas con estructuras conocidas. Estas mediciones pueden proporcionar un útil de control de calidad de la metodología y permitirá evaluar la precisión de los parámetros de adquisición mediante la comparación de los valores de Ω teóricos y medidos. El teórico áreas de corte transversal se puede calcular a partir de la estructura cristalina coordenadas utilizando el software MOBCAL 15,16, que es un software de código abierto basado en FORTRAN que permite la edición de código de acuerdo a las necesidades del operador. Para el funcionamiento de estos cálculos se requiere de modificar el programa de tal manera que el número de cálculos iterativos realizados por la estructura de entrada es mayor y que los archivos de coordenadas que contiene gran número de átomos son aceptados 1. Una estrategia de IM-MS para la definición de los arreglos topológicos de las subunidades en las asambleas de varios componentes se ha propuesto recientemente 4,6. El método consiste en la vigilancia de las vías de la disociación de las asambleas de proteínas a componentes más pequeños. Esta disociación se logra a través del ajuste de las condiciones de control en fase de solución, lo que da lugar a una distribución de subcomplejos reflejo de los &quot;bloques constructores&quot; de las asambleas. La medición simultánea de los valores de Ω tanto del complejo intacto y productos desmontaje genera restricciones estructurales que se utilizan para el cálculo de los modelos topológicos de los complejos de proteínas. El supuesto básico que subyace a esta metodología es que la subcomplejos generado conservar su lengua materna-como confirmaciones, de hecho, estudios recientes han demostrado que la estructura de la solución de los productos de desmontaje se mantiene y no hay reorganización importante en cualquiera de las soluciones o las fases de gas se han producido 4,6. El último paso en la asignación de la estructura cuaternaria de iones de proteína de fase de gas del complejo es conveniente los valores de la sección transversal de colisión con los modelos generados por computadora. Métodos de modelización, se emplean con el fin de explorar las diferentes disposiciones posibles topológica de subunidades y su in silico valores Ω se calculan y se comparan con los experimentales. Actualmente, sólo un pocos métodos computacionales se utilizan, como el spheretype de grano grueso, método que se aproxima el diámetro de las subunidades de 1,8. En general, este campo está todavía en sus primeros años de vida y el desarrollo se requiere para hacer este planteamiento genérico, y aplicable a una amplia gama de complejos.

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

De movilidad de iones (IM) es un método que mide el tiempo que tarda un ion de viajar a través de una célula de presión bajo la influencia de un campo eléctrico débil. La velocidad a la que los iones atraviesan la región de la deriva depende de su tamaño: los iones de gran experimentará un mayor número de colisiones con el gas inerte de fondo (por lo general N 2) y por lo tanto viajan más lentamente a través del dispositivo de mensajería instantánea que los iones que forman una pequeña sección transversal. En general, el tiempo necesario para que los iones a emigrar cuando la fase de gas denso que los separa, de acuerdo a la colisión de la sección transversal (Ω). Recientemente, la espectrometría de IM fue acoplada a espectrometría de masas y una de onda (la onda T) SYNAPT de movilidad de iones espectrómetro de masas (IM-MS) fue puesto en libertad. La integración de la espectrometría de masas con la movilidad iónica permite una nueva dimensión de la separación de la muestra y la definición, dando un espectro en tres dimensiones (masa de tiempo de carga, la intensidad y la deriva). Esta técnica de separación permite que el solapamiento espectral a disminuir, y permite la resolución de complejos heterogéneos con masas muy similares, o las relaciones masa-carga, pero diferentes momentos a la deriva. Por otra parte, las mediciones de tiempo deriva proporcionar una capa importante de la información estructural, ya que Ω tiene que ver con la forma general y la topología de los iones. La correlación entre los valores de tiempo medido a la deriva y Ω se calcula mediante una curva de calibración generada a partir de las proteínas con calibrador definido secciones 1. El poder del enfoque de IM-MS radica en su capacidad de definir el embalaje de la subunidad y la forma general de las asambleas de proteínas en concentraciones micromolar, y un corto condiciones fisiológicas. Varios estudios recientes de mensajería instantánea de ambas proteínas individuales 2,3 y no covalentes complejos de proteínas 4-9, logrado demostrar que la estructura de proteínas cuaternario se mantiene en la fase de gas, y destacó el potencial de este enfoque en el estudio de las cadenas proteicas de la geometría desconocida . Aquí, le ofrecemos una descripción detallada de IMS-MS análisis de complejos de proteínas utilizando el SYNAPT (cuadrupolo-Ion de movilidad en tiempo de vuelo) instrumento HDMS (Waters Ltd, el único comercial IM-MS instrumento actualmente disponible) 10. Se describen los pasos básicos de la optimización, la calibración de las secciones transversales de colisión, y los métodos para el procesamiento e interpretación de datos. El último paso del protocolo describe los métodos para calcular valores teóricos Ω. En general, el protocolo no pretende cubrir todos los aspectos de la IM-MS caracterización de conjuntos de proteínas, sino que su objetivo es introducir a los aspectos prácticos del método a los nuevos investigadores en el campo.

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

De movilidad de iones-espectrometría de masas es una emergente tecnología de fase gaseosa, que separa los iones, sobre la base de su colisión sección transversal y la masa. El método proporciona información en tres dimensiones de la topología general y la forma de complejos de proteínas. Aquí, se describe un procedimiento básico para elaborar un instrumento normativo y la optimización, la calibración de los tiempos de desplazamiento, y la interpretación de los datos.

De la onda T de movilidad de iones-espectrometría de masas: Basic Procedimientos experimentales para el análisis de proteínas complejas

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 vistas.  Weizmann Institute of Science. De movilidad de iones-espectrometría de masas es una emergente tecnología de fase gaseosa, que separa los iones, sobre la base de su colisión sección transversal y la masa. El método proporciona información en tres dimensiones de la topología general y la forma de complejos de proteínas. Aquí, se describe un procedimiento básico para elaborar un instrumento normativo y la optimización, la calibración de los tiempos de desplazamiento, y la interpretación de lo...

Video: 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 preparación de la muestra: el método de ultra capa fina

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

Investigación

Biología

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.

Cáncer de Borealis Stomatogastric disección del sistema nervioso

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).

La disección bruto del estómago de la langosta, 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.

No invasiva En Vivo Pequeños Animales MRI y MRS: Procedimientos básicos Experimental

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.

El análisis de grandes complejos de proteínas por espectrometría de masas estructurales

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.

Mutagénesis y análisis funcional de los canales iónicos heteróloga se expresa en células de mamífero

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.

Extracción de sangre para análisis bioquímicos en adultos de pez cebra

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.

Básico<em&gt; Caenorhabditis elegans</em&gt; Métodos de sincronización y de observación

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.

El Protein Complex Plataforma de Producción MultiBac en el EMBL

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.

El Enfoque ChroP Combina chip y la espectrometría de masas para diseccionar Locus específicos proteómicos Paisajes de la cromatina

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