우리는 프랭크 에르빈, 로버타 Palmour 및 영장류 작품들의 지속적인 지원 세인트 키츠, 서인도 제도에있는 Behavioural 과학 재단 연구소의 직원에게 감사하고 있습니다. 보건 연구 (AC)과 국립 공학 및 캐나다 (MP)의 연구위원회의 캐나다 연구소에서 운영 보조금 -이 작품은 깨지기 쉬운 X 리서치 SZ 캐나다의 재단 (FXRFC)와의 화목에 의해 지원되었다.

Immunohistochemisty 다양한 실험 동물 모델의 두뇌에있는 단백질 표현을 특성화를 위해 가장 널리 사용되는 기법 중 하나입니다. 유사한 두뇌 크기와 설치류 및 기타 일반적인 실험 모델의 두뇌에 체계적인 immunohistochemical 절차를 수행하기 위해 상대적으로 쉽습니다. 그러나, 전체 원숭이의 두뇌에 걸쳐 같은 면역 절차를 수행하는 방법에 대한 포괄적인 계정을 제공하는 우리의 지식에 게시된 작업은 없습니다. 어떤 다음은 다양한 대상 단백질의 대규모 immunohistochemical 감지에 대한 전체 원숭이 두뇌를 준비하는 방법에 대한 자세한 설명입니다. 이 작품은 상업 및 학문적 노력 사이의 협력의 결과로 떠오르고있다. 이와 같이 포함하고 sectioning 조직에 관한 세부 사항은 NSA의 독점적인 지식 남아 있습니다. 1 부 : 동물 치료 및 조직 준비 성인 vervet 원숭이 (Cercopithecus aethiops)에서 두뇌는 현재 프로토콜에 사용됩니다. 모든 절차는 생물 의학 연구 1 동물의 사용과 관리에 대한 애니멀 케어의 캐나다 협의회 (CCAC) 지침을 준수 실시하고 있습니다. <ol><li> 동물은 깊이 나트륨 pentobarbital (25 MG / kg, IV)와 완전히 exsanguinated 때까지 0.1 M PBS로 transcardially perfused의 과다로 euthanized 케타민을 하이드로 클로라이드 (10 MG / kg, IM)와 마취입니다. </li><li> 이것은 5 분 (~ 1리터)을 PBS에 4 % paraformaldehyde 솔루션옵니다. </li><li> 두뇌가 컨테이너의 하단에 싱크 ˚ C까지 externalized 두뇌는 0.02 %의 나트륨 azide를 포함한 등급 PBS 버퍼 자당 솔루션 (순서 10, 20, 30 %)에 위치 4에서 관리하고 있습니다. 두뇌가 냉동 sectioning를 겪습 때까지 솔루션은 수일마다 교체됩니다. </li><li> 그들의 독점 MultiBrain에 들어서는 ™ 삽입 및 냉동 sectioning 기술, 두뇌는 신경 과학 어소 (녹스빌, 테네시 NSA)로 전송됩니다. </li><li> 섹션이 제대로 지향되어야 (즉, 왼쪽, 오른쪽 방향)와 histological 처리가 완료된 후 다시 정렬 수 있도록 세븐 (7) 정렬 랜드마크가 포함 행렬에 배치됩니다. </li><li> 각각의 시리얼 부분이 블록에서 수집되기 전에 냉동 블록 디지털 블록 얼​​굴 사진입니다. 디지털 이미지뿐만 아니라이 두뇌의 부분은 나중에 각각 해부 및 histological지도 3D 재건에 사용될 수 있습니다. </li><li> 시리얼 자유 부동 코로나 섹션은 섹션 50 μm의의 두께, 전체 두뇌에서 수집됩니다. </li></ol> 2 부 : Histological 처리 섹션은 주어진 공간 간격으로 선택됩니다 (예, 500 μm의) 우리의 실험 목적을 위해 다음과 같은 항체로 처리되었습니다 연약한 - X 정신 지체 단백질 (FMRP, Chemicon, 테메큘라, CA), SMI32 (Sternberger Monoclonals 주식 회사; 볼티모어, MD), 그리고 NeuN (Chemicon, 테메큘라, CA). FMRP는 다량으로 정상과 순열 사업자 두뇌의 뉴런이 발견 세포질 단백질이다. NeuN (핵의 연결)는 구체적으로 대부분의 뉴런에 존재하며 핵의 연결, perikarya 일부 근위의 연결 프로세스 3 배포되는 DNA 바인딩 신경 세포 특정 단백질 NeuN를 인식합니다. SMI - 32 neurofilament 단백질 4 비 phosphorylated 에피토프을 인식 단클론 항체이다. <ol><li> 500 μm의의 간격, 약 140 섹션은 항체마다 사용됩니다. </li><li> 모든 incubations, 세척 및 얼룩 단계는 전문 얼룩 요리와 바구니 (; 녹스빌, 테네시 HistoTools)를 사용하여 가벼운 동요와 함께 진행하고 있습니다. </li><li> 섹션 먼저 항체 분자의 특이 현상이 자꾸 발생 배경 얼룩을 줄이기 위해 0.1 M PBS/0.3 % 트리톤 X - 100 (TX) / 60 분 5 % 정상 쉰 혈청 (NHS)이있는 솔루션에 incubated 수 있습니다. </li><li> 공급 업체의 지시에 따라, immunostaining FMRP에 대한 표시 섹션 항원 Unmasking 솔루션 (버링게임, CA 벡터 연구소)를 사용하여 항원 복구의 추가 단계를 받고있다. </li><li> 섹션은 다음 하룻밤과 0.1 M PBS/0.3 % TX / 3 % NHS를 (NeuN 항체에 대한 FMRP 및 SMI - 32과 1 / 2, 000에 대한 5분의 1, 000의 희석 요소로 포함하는 각각의 항체 솔루션에서 별도의 일 incubated 수 있습니다. </li><li> 1 /; 다음날은 섹션은 말에서 제기 biotinylated 방지 마우스 이차 항체에 실온에서 2 H (벡터 연구소에 대한 부화 다음 세척 용액 세 10 분 기간 (0.1 M PBS/0.3 % TX)에 씻어 아르 0.1 M PBS/0.3 % TX / 3 % NHS 1,000 희석). </li><li> 3 10 - 분 세척을 추가로 설정 후, 섹션은 실온에서 1 H에 대한 아비딘 - 비오틴 복합 양고추냉이 퍼옥시데이즈 복합 (벡터 연구소)의 솔루션에 배치됩니다. </li><li> 마지막으로, 세척 다른 설정 후, 섹션은 니켈 향상된 디아 10 분 대상이 아르minobenzidine (NI - DAB) 진한 파란색이 immunoreactive 뉴런 이내에 얼룩이 생성 반응. </li><li> histological 절차의 완료에, 부분은 젤라틴 - 코팅 유리 슬라이드에 탑재하고 있습니다. </li><li> 모든 섹션은 공기 건조 하룻밤되고 표준 절차에 따라 coverslipped. </li></ol> 파트 3 : 대표 결과 : 이 방법은 전체 원숭이 두뇌에 걸쳐 관심의 대상 단백질의 완전한 표현 프로필을 생산하고 있습니다. 여기 FMRP, NeuN과 같은 원숭이의 뇌에 SMI32 표현의 스냅샷을 제공하는 대표적인 코로나 섹션을 표시합니다. <img src="/files/ftp_upload/1286/1286fig1.png" alt="" /> 그림 1 : 스테 이닝 요리 (A)와 바구니 (B)가 면역 무료 - 부동 섹션의 대규모 일괄 처리에 사용. <img src="/files/ftp_upload/1286/1286fig2.png" alt="" /> 그림 2 : FMRP (A), NeuN (B)와 SMI32 (C) 항체에 대한 스테인드 vervet 원숭이 머리에서 대표 코로나 섹션.

<ol>
	<li>Olfert, E. D., Cross, B. M., McWilliam, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to the Care and Use of Experimental Animals. , (1993).</li><li>Devys, D., Lutz, Y., Rouyer, N., Bellocq, J. P., Mandel, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FMR-1+protein+is+cytoplasmic,+most+abundant+in+neurons+and+appears+normal+in+carriers+of+a+fragile+X+premutation.">The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation.</a> Nat Genet. 4, 335-340 (1993).</li><li>Mullen, R. J., Buck, C. R., Smith, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NeuN,+a+neuronal+specific+nuclear+protein+in+vertebrates.">NeuN, a neuronal specific nuclear protein in vertebrates.</a> Development. 116, 201-211 (1992).</li><li>Sternberger, L. A., Sternberger, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+distinguish+phosphorylated+and+nonphosphorylated+forms+of+neurofilaments+in+situ.">Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.</a> Proc Natl Acad Sci U S A. 80, 6126-6130 (1983).</li></ol>

The authors have nothing to disclose.

가능한 전체 원숭이의 뇌에 단백질의 대규모 감지하게이 절차에서 두 중요한 단계가 있습니다. 하나는 NSA의 독점적인 지식 남아 삽입과 sectioning 프로토콜입니다. 다른 하나는 HistoTools에서 제공하는 얼룩 요리와 바구니를 사용하는 것입니다. 후자는 주어진 시간에 많은 (~ 40) 섹션을 쉽고 빠르게 처리하실 수 있습니다. 또한 균일하게 뇌 전체 부분을 치료를위한 수단을 제공하고 과학적으로 소리 h...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>anti-FMRP monoclonal antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>MAB2160</td>
<td>Requires antigen retrieval in fixed tissue.</td>
</tr>
<tr>
<td>anti-NeuN monoclonal antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>MAB377</td>
<td>N/A</td>
</tr>
<tr>
<td>anti-Neurofilament H monoclonal antibody</td>
<td>&nbsp;</td>
<td>Sternberger Monoclonals Inc.</td>
<td>SMI32</td>
<td>N/A</td>
</tr>
<tr>
<td>Antigen Unmasking Solution</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>H-3300</td>
<td>N/A</td>
</tr>
<tr>
<td>Normal horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050122</td>
<td>500 mL</td>
</tr>
<tr>
<td>Biotinylated Anti-Mouse IgG (H+L), made in horse</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>BA-2000</td>
<td>&nbsp;</td>
<tr>
<td>VECTASTAIN ABC Kit (Standard)</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>PK-4000</td>
<td>1.5 mg</td>
</tr>
<tr>
<td>Staining dish for floating sections</td>
<td>&nbsp;</td>
<td>HistoTools</td>
<td>10009</td>
<td>12 x 65 mm</td>
</tr>
<tr>
<td>Staining transport basket</td>
<td>&nbsp;</td>
<td>HistoTools</td>
<td>10012</td>
<td>large 
(fits 12 x 65 mm dish)</td>
</tr>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>P8514B</td>
<td>N/A</td>
</tr>
<tr>
<td>DAB</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>AC11209-0050</td>
<td>N/A</td>
</tr>		</tbody>
		</table>
		</div>

batch immunostaining for large-scale protein detection in the whole monkey brain

Immunohistochemistry (IHC)는 대상 단백질의 검출에 가장 널리 사용되는 실험 기법 중 하나입니다<em&gt; 현장에서</em&gt;. 전체 두뇌를 통해 대상 단백질의 표현 패턴에 관한 질문은 설치류의 것과 작은 두뇌에 IHC를 사용하는 경우 답변을 상대적으로 쉽습니다. 그러나, 영장류의 것과 같은 대규모 뒤얽힌 두뇌에서 같은 질문에 대답하는 것은 도전의 번호를 보여줍니다. 여기서 우리는 어른 원숭이 두뇌의 대상 단백질의 면역을위한 체계적인 접근법을 제시한다. 이 방법은 신경 과학의 방법론 어소 (NSA)뿐만 아니라 자유 부동 섹션의 배치 얼룩을 위해 특별히 개발된 도구를 포함하고 sectioning 조직에 의존합니다. 그것은 특정 간격으로, 전체 두뇌를 대표하는, 섹션의 집합의 유니폼을 얼룩을 초래합니다. 그 결과 얼룩진 부분은 단백질 수준, 특정 단백질을 표현 뉴런의 인구를 측정하기 위해 분석 절차의 다양한 받게 수 있습니다.

Watch this Scientific Journal Video about Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain at JoVE.com

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

전체 영장류 두뇌를 통해 대상 단백질의 대규모 면역이 포함 소설 조직을 고용하고 주어진 시간에 여러 자유 부동 섹션의 배치 얼룩을위한 창조적인 장치를 이용하여 결합 방법을 sectioning에서 가능합니다.

전체 몽키 브레인에서 대규모 단백질 탐지에 대한 배치 Immunostaining

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

batch-immunostaining-for-large-scale-protein-detection-whole-monkey

Research

JoVE Journal

Biology

12.6K 조회수.  Montreal Neurological Institute. 전체 영장류 두뇌를 통해 대상 단백질의 대규모 면역이 포함 소설 조직을 고용하고 주어진 시간에 여러 자유 부동 섹션의 배치 얼룩을위한 창조적인 장치를 이용하여 결합 방법을 sectioning에서 가능합니다.

비디오: Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Metagenomic 도서관 대규모 화면

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

연구

생물학

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

단백질 Ubiquitination의 감지

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

의 정화 M. magneticum 스트레인 AMB - 1 Magnetosome 관련 단백질 MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

대규모 Zebrafish 기반 생체내에서 작은 분자 화면

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

대규모 SNP 유전자형 응용 프로그램에 대한 Infinium 분석

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

민감한, 대규모 양적 대사 체학을위한 전략

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

생물 발광 공명 에너지 전달을 이용하여 살아있는 세포에서 단백질 - 단백질 상호 작용을 조사

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY : 효모 성장의 다 변수 정량화를위한 ​​대규모 방법

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

헬리안투스 아누스의 대규모 열화상 스크리닝에 의한 새로운 식물 증발 규제 기관 식별

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (&#62;100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

This paper describes a semiautomated, microscaled ChIP-Seq protocol of DNA regions associated with a specific histone modification (H3K27ac) using a ChIP liquid-handler platform, followed by library preparation using tagmentation. The procedure includes control assessment for quality and quantity purposes and can be adopted to other histone modifications or transcription factors.

대규모 후생유전학 연구를 위한 반자동 ChIP-Seq 절차

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...