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. 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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

Nanyang Technological University

Research

JoVE Journal

Biology

24.4K 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

Homarus Americanus Stomatogastric Nervous System Dissection

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the activity of interest and are accessible and amenable to experimental methods. The stomatogastric nervous system (STNS) of the American lobster (Homarus americanus; also know was the Atlantic or Maine lobster) has been established as a model system for studying rhythm generating networks and neuromodulation of networks. The STNS consists of 3 anterior ganglia (2 commissural ganglia and an oesophageal ganglion), containing modulatory neurons that project centrally to the stomatogastric ganglion (STG). The STG contains approximately 30 neurons that comprise two central pattern generating networks, the pyloric and gastric networks that underlie feeding behaviors in crustaceans1,2. While it is possible to study this system in vivo3, the STNS continues to produce its rhythmic activity when isolated in vitro. Physical isolation of the STNS in a dish allows for easy access to the somata in the ganglia for intracellular electrophysiological recordings and to the nerves of the STNS for extracellular recordings. Isolating the STNS is a two-part process. The first part, dissecting the stomach from the animal, is described in an accompanying video article4. In this video article, fine dissection techniques are used to isolate the STNS from the stomach. This procedure results in a nervous system preparation that is available for electrophysiological recordings.

Homarus Americanus Stomatogastric Nervous System Dissection

We describe the fine dissection of the stomatogastric nervous system from the stomach of the American lobster (Homarus americanus).

Homarus Americanus Stomatogastric 신경계의 해부

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the ...

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 ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

액상 복구와 분자량 기반 분류에 대한 GELFREE 8100 분획화 시스템을 사용하여

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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 ...

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 ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...