우리가 그들의 기술 출자를위한 Servane Contal와 보리스 Untereiner 감사드립니다. 우리는 FNR &quot;FUTOX&quot;프로젝트의 지원을 인정합니다.

프로토콜은 완성 16 일 필요합니다. 기간은 그림 1에 자세히 있습니다. 버퍼 준비 세부 <ul><li> 용해 완충액 (샘플 당 3mL)를 준비. </li><li> 세탁 버퍼 (샘플 당 50mL)의 준비. 이 버퍼는 미리 냉장 및 저장 -20 ° C에서 냉동실에 대한 분석까지 전체 절차를 수행하는 동안 얼음에 보관해야합니다. </li><li> 강수량 버퍼 (샘플 당 20mL)의 준비. 전체 절차를 수행하는 동안 필요한 얼음에 보관 때까지 버퍼는 냉동실에 -20 ° C에서 사전 냉장 및 저장해야합니다. </li><li> 작품은 우선적으로 4 추위 챔버로 진행되어야 ° C. </li></ul> 곰팡이 종 proteomic 분석에 사용되는 세 가지 변종들은 형태학의 기능과 번역 - 연신율 팩터 알파 - 1 분석을 바탕으로 Fusarium의 graminearum로 분류되었다 (오도넬 외., 1998) 이 변종은 CRP - 가브리엘 Lippmann 컬렉션에 속한 유지와에 저장된 - 80 ° C 15 %의 글리세롤. mycelia의 준비 곰팡이는 25 ° C 빛과 어둠의 기간을 번갈아 가며 각 12 시간에서 4 일간 V8에서 재배되었다. 문화는 살균 칼날과 이러한 문화의 조각 독소 - 유도 미디어 100 ML를 포함 250 ML Erlenmeyer flasks의를 예방하는 데 사용되었다를 사용하여 조각난되었습니다. Mycelia 10 일 후에 수확되었고 살균 필터와 문화를 필터링하여 중간에서 분리되었다. Mycelia는 중간 잔류물 및 기타 화합물을 제거하는 살균 물을 3 번 씻어했다. 그러면 즉시 액체 질소를 추가하여 세포를 동결하고 -80에서 샘플을 보관 ° C. 단백질 추출에 대한 일반적인 설명 단백질 샘플은 그림 3A와 3B에서 설명한 방법을 사용하여 추출되었다. Fusarium 샘플은 액화 질소에 지상되었고 분말 10 ML 테플론 튜브에 수집되었다. 샘플은 다음, 용해 버퍼로 30 분 incubated 10 분 끓인하고, 실온 (15 분 1만2천그램)에 2 번 centrifuged했다. supernatants는 하루 강수량 버퍼와 -20 ° C에서 수집된와 incubated했다. 4 원심 분리 후 ° C 3만그램 45 분 간은 시켰던 단백질의 알약이 DTT를 포함하는 차가운 아세톤과 함께 3 번 씻어했다. 마지막으로, 알약은 공기 건조되었으며 단백질은 8 산도 조정, 라벨 버퍼에 resolubilized되었습니다. 30 ML의 하위 샘플은 총 단백질 양을 정함 위해 제거된 나머지 뜨는은 단백질 전기 영동까지 -20 ° C에서 보관되었다. <img src="/files/ftp_upload/1690/1690fig1.jpg" alt="그림 1" /> 그림 1. 절차의 타임 라인. <img src="/files/ftp_upload/1690/1690fig2.jpg" alt="그림 2" /> 그림 2. 여러 연산자 샘플 처리를위한 제도. <img src="/files/ftp_upload/1690/1690fig3atmb.jpg" alt="그림 3A" border="0" /> 그림 3A. <img src="/files/ftp_upload/1690/1690fig3btmb.jpg" alt="그림 3B" border="0" /> 그림 3B는하시기 바랍니다. <a href="/files/ftp_upload/1690/1690fig3a.jpg">여기를 클릭하십시오</a> 더 큰 그림 3A 버전, 또는 ​​볼 수 <a href="/files/ftp_upload/1690/1690fig3b.jpg">는 여기에</a> 그림 3B의 더 큰 버전을 위해. 그림 3A - B 단백질 추출 절차 흐름 차트 (A : 첫날, B : 두번째 날).. <img src="/files/ftp_upload/1690/1690fig4tmb.jpg" alt="그림 4" border="0" /> 그림 4. 단백질 추출물 1D 그림. 단백질은 용암 퍼플 물들일 있던 젤의 끝에 파란색 완벽하게 마이 그 레이션까지 실행했다. 하시기 바랍니다 <a href="/files/ftp_upload/1690/1690fig4.jpg">여기를 클릭</a> 그림 4의 더 큰 버전을 볼 수 있습니다.

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곰팡이 생물 도메인 내에서 proteomic 접근법에 최근 관심이 기술을합니다 (김 외., 2007 년 검토)를 사용하는 출판물의 증가 수를되었다. 단백질 추출을위한 방법은 추출 절차의 조합에 의존하고, 그들은 종종 기뻐서 방법론 섹션에서 설명하지 않고 잠재적인 문제 해결 방법을 다루는의 전문 지식을 요구하고 있습니다. 샘플 및 데이터 처리를위한 표준을 설정 기타 &quot;omics&quot;접근에 대해서는 것은 신뢰할 수있는 과학적 정보를 생성하기 위해서는 필수적입니다. 또한 샘플 및 조작의 절차가 적절하게 다른 &quot;omics&quot;실험간에 공유 결과 해석을 수 있도록 설명해야합니다. 이것은 완전히 게놈, proteomics, metabolomics에 간 데이터 마이닝의 잠재력을 활용을 위해 필수적인 것입니다 ... (모리슨 외., 2006). 실험의 모든 측면 (겔 전기 영동, 질량 분광법, 분자 상호 작용, 단백질 수정, Proteomics의 정보학, 샘플 처리)에 대처하기 위해서는 proteomic 실험에 대한 최소 정보는 초안되었으며 작업 그룹이 설립되었습니다. 현재에는 정의된 정보는 샘플 처리 절차에 사용할 수 없습니다. (테일러 외., 2007) MIAPE 지침의 골격 내에서 표준화를 높일 수 있습니다 절차를 구현하기 위해, proteomic 연구의 최종 품질을 결정하는 단백질 추출 절차의 중요성을 감안할 때, 우리는 샘플을 상세하게 비디오 프로토콜의 사용을 제안 처리. 실험의 비디오 설명은 &quot;omics&quot;실험 (Pasquali, 2007)보다 재현성을 초래할 수 샘플 처리에 대한 정보의 수를 늘리기 위해 크게 기여할 수 있습니다. 사실, 실험실에 걸쳐 재현성은 proteomics 결과 (http://www.fixingproteomics.org)의 유효성을 보장하기 위해 기본적인 요구 사항입니다. 다른 instrumentations과 다른 연산자 샘플을 조작 : 크로스 실험실 재현성은 두 요인에 의해 영향을받습니다. 설명 프로토콜에서, 우리는 수행하기 위해 제안 생물 학적 데이터 (즉, 결과의 기술 변화가 고려됩니다)의 신뢰성을 높이기 위해 여러 개의 연산자 (그림 2 및 비디오)를 포함하는 복제합니다. 단백질 추출에 대해 설명한 절차는 SDS와 난방을 기반으로합니다. 이것은 이전에 단백질의 좋은 순도와 수량 (브리지 1996) 보장하는 표시했다. SDS는 세포 벽, 소수성 단백질을 해체하고 oligomers 형성 단백질의 중단 석출을 방지 끓고와 결합. 비등은 프로 테아제의 불활 성화도 있습니다. 같은 효과는 EDTA (에틸렌 다이아 민 테트라 초산), PMSF하고 완전한 미니 테아제 억제제에 의해도 생산합니다. DTT는과 단백질 solubilization을 촉진 단백질 사이의 이황화 다리를 제거합니다. IEF 전에 SDS를 제거하기 위해 단백질은 아세톤 / TCA / DTT에 의해 시켰던 있습니다. 또한, 아세톤 / TCA / DTT 강수량이 화합물이 존재할 때 이러한 lipids, 핵산, 염분 또는 / 및 페놀 수지 carbon 화합물 같은 오염 물질을 제거하실 수 있습니다. SDS와 마찬가지로,이 분자는 IEF 동안 좋은 마이 그 레이션되지 않습니다. 이 강수량에 따라, 그것이 IEF를 방해할 수있는 TCA를 제거하기 위해 아세톤 / DTT에 의해 시켰던 단백질을 씻어 필요합니다. 좋은 샘플 준비는 좋은 결과의 핵심입니다. 이를 위해, 분말 무료 장갑 작업과 좋은 실험실 관행을 존중 환경에서 단백질의 오염을 방지하는 것도 중요 그것이다. 보기의 기술적 관점에서 설명하는 프로토콜 내에서 단백질의 충분한 양을과 정결을 얻기위한 가장 중요한 단계는 두 단계입니다. 먼저, 연삭 세포의 완전한 파괴는 단백질을 공개하는 근본이다 단계, 둘째, 총 단백질의 정화, lipids, DNA와 단백질의 마이 그 레이션을 방해할 수있는 다른 오염 물질의 대부분의 제거를 포함한 위상.

미디어 솔루션 PDA : 39g 포테이토 덱스 트로 오스 아가르 (Difco), 1 L 증류수. V8의 한천 : 200 ML V8 (캠벨, 미국), 2g CaCO3 (시그마), 16g 아가르 (DIFCO), 800 ML 증류수 독소의 유도 매체 : 1g K 2 HPO 4 0.5g KCl, 0.5g MgSO 4 7H 2 O, 10 MG FeEDTA, 2g L 글루탐산, 증류수 1L에 10g의 자당. 용해 완충액 : <table border="1"><tr><td></td><td> 100mL </td><td> 최종 농도 </td><td></td></tr><tr><td> 트리스 - HCL pH8 </td><td> 5mL </td><td> 50mM </td><td> 1M </td></tr><tr><td> SDS </td><td> 2g 또는 10mL </td><td> 2퍼센트 </td><td> 20% </td></tr><tr><td> DTT </td><td> 500mL </td><td> 10mM </td><td> 2M </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 20mL </td><td> 0.1mM </td><td> 0.5M </td></tr><tr><td> PMSF </td><td> 200mL </td><td></td><td></td></tr><tr><td> 완료 미니 테아제 억제제 </td><td> 1 정제 </td><td></td><td></td></tr><tr><td> 물 </td><td> Qsp </td><td></td><td></td></tr></table> 강수량 버퍼 : <table border="1"><tr><td></td><td> 100mL </td><td> 최종 농도 </td></tr><tr><td> TCA </td><td> 20mL </td><td> 20% </td></tr><tr><td> DTT </td><td> 0.1mL </td><td> 0.1 % </td></tr><tr><td> Aceton </td><td> Qsp </td><td></td></tr></table> 버퍼 워싱 : <table border="1"><tr><td></td><td> 100mL </td><td> 최종 농도 </td></tr><tr><td> DTT </td><td> 0.1mL </td><td> 0.1 % </td></tr><tr><td> Aceton </td><td> 100mL </td><td></td></tr></table> Labelling 버퍼 (° C 작은 aliquots에서 -18에 저장) <table border="1"><tr><td></td><td> 1mL </td><td> 최종 농도 </td></tr><tr><td> 요소 </td><td> 140 MG </td><td> 은 7m </td></tr><tr><td> 티오 우레아 </td><td> 50 MG </td><td> 2M </td></tr><tr><td> 챕스 </td><td> 40 MG </td><td> 4퍼센트 </td></tr><tr><td> 트리스 </td><td> 30 μL </td><td> 30 MM </td></tr><tr><td> 물 </td><td> 1 ML </td><td></td></tr></table>

toxin induction and protein extraction from fusarium spp. cultures for proteomic studies

Fusaria 다른 독소를 생산할 수 filamentous 곰팡이 있습니다. 이러한 deoxynivalenol, nivalenol, T2, zearelenone, fusaric 산, moniliformin 등 Fusarium의 mycotoxins .. 인간과 동물 모두 건강에 악영향을하고 일부는 pathogenicity 요인으로 간주됩니다. Proteomic 연구 Fusaria 잠재적인 병원성 요인 (종이 외., 2007, Houterman 외., 2007) 식별을위한 독소 생산 메커니즘 (테일러 외., 2008)뿐만 아니라 해독 효과로했다. 그것은 실험, 종자 및 연구소 간의 단백질 발현에있는 진실의 차이에 의존하기 위해 proteomic 연구 간의 비교에 대한 신뢰할 수있는 방법을 확립하는 것이 기본이된다. 설명되는 절차는 두 가지 방법에 의해 proteomic 절차의 표준화 수준 증가에 기여한다. 촬영 프로토콜은 정확하게 표현할 수있는 세부 수준을 향상하는 데 사용됩니다. 또한, 생물 학적 처리 표준 절차의 가용성 추출 절차의 기술 재현성 내에서 또한 계정에 인간의 요소를 복용 데이터의 높은 견고을 보장해야 복제합니다. 문화와 단백질 추출 (그림 1)의 두 가지 일 십사일 : 설명 프로토콜은 완성 16 일 필요합니다. 간단히, Fusarium의 종자는 4 일 동안 고체 매체에 재배되며 그들은 다음 수동 조각과 10 일 동안 수정된 독소의 유도 매체 (Jiao 외, 2008.)으로 전송됩니다. 균사체는 Miracloth 계층을 통해 여과에 의해 수집됩니다. 연마는 감기 챔버에서 수행됩니다. 추출을 수행 다른 연산자는 계정으로 기술 변화 (그림 2)로 인해 편견을하기 위해 (N = 3)를 복제합니다. 추출로 테일러 외에 설명된 SDS / DTT 버퍼를 기반으로했다. (2008) 약간 수정. 총 단백질 추출 Aceton / TCA / DTT 버퍼 야간 및 DTT / 아세톤 세척 (그림 3A, 3B)를 사용하여 단백질의 침전 과정을 필요합니다. 단백질은 결국 단백질 분류 버퍼 resolubilized 및 계량했다. 추출 결과는 2D 젤 (IEF / SDS - PAGE)로 진행하기 전에, 1D 젤 (그림 4, SDS - PAGE)에 시각되었습니다. 동일한 절차는 다른 성장 미디어 및 기타 filamentous 곰팡이 (마일즈 외., 2007)에서 proteomic 분석에 대해 적용할 수 있습니다.

Watch this Scientific Journal Video about Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies at JoVE.com

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

곰팡이 종의 proteomic 분석을위한 단백질 추출 proteomic 실험 (MIAPE) 지침에 대한 최소한의 정보에 따라 달성하는 표준화의 높은 수준이 필요합니다. 우리는에서 독소 유도와 단백질 추출하는 동안 실험 바이어스를 최소화하는 절차를 포함하는 비디오 프로토콜을 제시<em&gt; Fusarium 종.</em

에서 독소 유도와 단백질 추출 Fusarium 종.</emProteomic 연구> 문화

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

toxin-induction-protein-extraction-from-fusarium-spp-cultures-for

Nanyang Technological University

Research

JoVE Journal

Biology

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

16.9K 조회수.  Centre de Recherche Public-Gabriel Lippmann. 곰팡이 종의 proteomic 분석을위한 단백질 추출 proteomic 실험 (MIAPE) 지침에 대한 최소한의 정보에 따라 달성하는 표준화의 높은 수준이 필요합니다. 우리는에서 독소 유도와 단백질 추출하는 동안 실험 바이어스를 최소화하는 절차를 포함하는 비디오 프로토콜을 제시 Fusarium 종.

비디오: Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

RNA 또는 특정 뇌 영역에서 단백질 추출에 대한 쥐 두뇌의 마이크로 해부

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

연구

생물학

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

단백질의 전기 영동 분리

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Interview: Protein Folding and Studies of Neurodegenerative Diseases

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

인터뷰 : 단백질 폴딩과 Neurodegenerative 질병의 연구

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the ...

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

신경 세포와 Glia 개발 연구 랫 두뇌 집계 문화의 준비

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

자동 Proteomic 처리를위한 디지털 Microfluidics

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

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 ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

유전과 Epigenetic 분석을위한 파라핀 임베디드 재료에서 DNA 추출

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Sexual Development and Ascospore Discharge in Fusarium graminearum

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished).
The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay.
F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Sexual Development and Ascospore Discharge in Fusarium graminearum

Sexual crosses and isolation of recombinant progeny are important research tools for the filamentous fungus, Fusarium graminearum, The techniques necessary successfully carry out these processes are presented.

의 성적 개발 및 Ascospore 방전 Fusarium graminearum</em

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

인간의 기본 섬유 아세포 배양에서 순진 성인 줄기 세포 분리

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

비교 단백체 분석을위한 신진 대사 라벨링 및 막 분별<em&gt; 애기 장대</em&gt; 서스펜션 세포 배양

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

에서 독소 유도와 단백질 추출 Fusarium 종. 문화