Name: dynamic proteomic and mirna analysis of polysomes from isolated mouse heart after langendorff perfusion
Uploaded: 2018-08-29T13:00:00
Description: Watch this Scientific Journal Video about Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion at JoVE.com

NIH P01 HL112730 (걸 레, JVE), NIH R01 HL132075 (걸 레, JVE), 바브라 스 트라이 샌드 여자의 심장 센터 (걸 레, JVE), 도로시와 E. 필립 리옹 분자 심장 (걸 레)에서 자, 에리카 글 레이저 부여 여성의 심장 건강 (JVE)에 체코 과학 아카데미의의 자 제도적 지원 RVO: 68081715 (MS)

모든 동물 연구 기관 지침에 따라 수행 하 고 기관 동물 관리 및 사용 위원회의 삼목 시 나이 의료 센터에 의해 승인 했다.
1. Langendorff 마우스 심장의 관류
<ol><li>
국 소 빈 혈과 reperfusion 마우스 마음의 Langendorff 관류
	<ol>
<li>관리 복 pentobarbital 나트륨 70 mg/kg 성인 마우스 (8 주 오래 된, 남성, C57BL6/j). 핀치를 발 끝에 철수의 부족에 의해 깊은 마 취를 확인 합니다.</li...

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Polysome 프로필 분석 특정 mRNA 또는 전체 transcriptome6,7의 변환 상태를 분석 하 여 단백질 번역의 연구에 대 한 수 있습니다. 그것은 또한 큰 도움이 현지 번역 synaptosomes8같은 공부 될 필요가 있을 때입니다. 전통적으로,이 방법은 모노-및 polyribosomes 및 자당 그라디언트는 수와 결합 게놈 또는 의도 된 결과6,<sup clas...

마음은 (I) 허 혈과 reperfusion 동적 방식에서 (R)의 부상에 응답합니다. 그러나, 응답 하는 동안 단백질 합성의 급성 변화에 약간의 통찰력이 있다. 이 해결 하기 위해 우리가 했다 polysome1 단백질 풍부 리보솜 및 polysomes, cytosol에서 변환 규제 요인의 재분배를 반영 하는 변화를 식별 하기 위해 프로 파일링의 기초가 튼튼한 방법의 장점 및 새로 합성된 단백질 (NSPs)에 증가. 나의 설정에서 / R, 새로운 mRNAs2;의 전사와 일치 하지 않는 시간에 발생 하는 새로운 단백질 합성의 증가 또한, mRNA 식 수준 및 단백질 풍부 사이 부조화 보고3되었습니다. 이러한 이유로, 우리는 동적 프로테옴에서 변경 내용을 분석 하는 단백질 번역에 의해 반영 하기로 결정 했다. 이렇게 하려면, 우리는 polysome 분수에서 mRNA를 계량 하 고 polysome 분수의 단백질 구성을 분석. 마지막으로, microRNAs (미르) 번역에 대 한 mRNAs의 조절 및 단백질 번역4,5의 효율을 방해할 수 있습니다, 때문에 우리 검사 polysome 분수에 미르의 배포에 초점을 맞추고는 내가 응답 / R.
우리는 지속적인 관류, 그리고 국 소 빈 혈 reperfusion의 30 분 뒤의 30 분 30 분 글로벌 아니 흐름 허 혈 후의 기저 상태에서 수확 조직과 격리 마우스 Langendorff 관류 모델을 사용 하기로 결정 했습니다. 우리 심장 조직의 solubilized 그리고 자당 기온 변화도 통해 polysomes를 분리 proteomic 분석 및 선택적 검출 mRNAs와 PCR 및 microarray, miRNAs의 각각 다음. 방법의이 조합은 mRNA, 미르, NSPs의 동시 검출 뿐만 아니라 nontranslating 분수 사이 규제 단백질, 미르, 그리고 mRNA의 재배포 가능 동적 프로테옴을 이해 하는 강력한 접근 방식을 나타냅니다. 낮은 효율 polysomes, 그리고 높은 효율 polysomes ( 그림 1참조). 이 프로세스의 동적 규칙에 eIF2α 또는 mTOR 등 규제 요인의 인 산화의 더 분석 하 여 연장 됩니다. 이러한 개별 단계 이제 자세히 설명 합니다.

<table>
	<tbody>
		<tr>
			<td>Pentobarbital</td>
			<td>Vortech Phamaceuticals</td>
			<td>9373</td>
			<td>for euthansia</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sagent</td>
			<td>103424</td>
			<td>used in langendorff preparation</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Fine Science Tools</td>
			<td>91110-10</td>
			<td>used to hang the heart</td>
		</tr>
		<tr>
			<td>Langendorff system</td>
			<td>Radnoti + home made</td>
			<td>n/a</td>
			<td>A &#39;four heart&#39; system consisting of custom blown glass, tubing and water baths</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653-5KG</td>
			<td>Krebs buffer and Sucrose gradient</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td>Krebs buffer and Lysis buffer</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P-5504</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M7774-500G</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G5767</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016-500G</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>Sucrose powder</td>
			<td>Sigma</td>
			<td>S0389-1KG</td>
			<td>Sucrose gradient</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>208337</td>
			<td>Sucrose gradient and Lysis buffer</td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td>Sucrose gradient and Lysis buffer</td>
		</tr>
		<tr>
			<td>Xylene Cyanole</td>
			<td>Sigma</td>
			<td>X-4126</td>
			<td>Sucrose gradient</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma-aldrich</td>
			<td>239763</td>
			<td>Sucrose gradient and Lysis buffer</td>
		</tr>
		<tr>
			<td>RNaseOUT</td>
			<td>Life Technologies</td>
			<td>C00019</td>
			<td>RNAse inhibitor for Lysis buffer</td>
		</tr>
		<tr>
			<td>Igepal CA-360 (NP40)</td>
			<td>Sigma</td>
			<td>I3021</td>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail tablets, EDTA free</td>
			<td>Roche</td>
			<td>5056489001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube, Thinwall, Ultra-Clear, 13.2 mL, 14 x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td>LE-80K</td>
			<td>Ultracentrifugation of the gradients</td>
		</tr>
		<tr>
			<td>Rotor</td>
			<td>Beckman</td>
			<td>SW41</td>
			<td>Ultracentrifugation of the gradients</td>
		</tr>
		<tr>
			<td>Biologic LP (pump)</td>
			<td>Biorad</td>
			<td>731-8300</td>
			<td>Fractionation of the gradients</td>
		</tr>
		<tr>
			<td>BioFrac</td>
			<td>Biorad</td>
			<td>741-0002</td>
			<td>Fractionation of the gradients</td>
		</tr>
		<tr>
			<td>Eppendorf RNA/DNA LoBind microcentrifuge tubes, 2 mL tube</td>
			<td>Sigma</td>
			<td>Z666513-100EA</td>
			<td>Gradient fraction and RNA extraction</td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Life technologies</td>
			<td>AM9738</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Luciferase Control RNA</td>
			<td>Promega</td>
			<td>L4561</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C606-4</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Glycogen, RNA grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0551</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma</td>
			<td>I9516</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>E7023-1L</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>BioRad</td>
			<td>170-8891</td>
			<td>Reverse transcription</td>
		</tr>
		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>BioRad</td>
			<td>175-5122</td>
			<td>Quantative PCR</td>
		</tr>
		<tr>
			<td>miRNeasy Micro Kit (50)</td>
			<td>Qiagen</td>
			<td>217084</td>
			<td>Kit for total RNA isolation</td>
		</tr>
		<tr>
			<td>miScript II RT Kit (50)</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td>Kit for miRNA reverse transcription</td>
		</tr>
		<tr>
			<td>miScript Sybr Green PCR Kit (200)</td>
			<td>Qiagen</td>
			<td>218073</td>
			<td>Kit for real-time PCR expression analysis of miRNAs</td>
		</tr>
		<tr>
			<td>Centrifuge 5424R</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For centrifugation of 1.5ml or 2.0ml tubes at different temperatures. Max speed - 21130g</td>
		</tr>
		<tr>
			<td>Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For real-time PCR plate centrifugation at different temperatures. Max speed - 2039g</td>
		</tr>
		<tr>
			<td>My Cycler Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>For reverse transcription</td>
		</tr>
		<tr>
			<td>CFX96 Real-Time System/C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>For real-time PCR analysis</td>
		</tr>
		<tr>
			<td>miRNeasy Serum/Plasma Spike-in Control</td>
			<td>Qiagen</td>
			<td>219610</td>
			<td>For quality control of RNA isolation</td>
		</tr>
		<tr>
			<td>Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, green/clear</td>
			<td>Bio-Rad</td>
			<td>HSP9641</td>
			<td>For real-time PCR analysis</td>
		</tr>
		<tr>
			<td>Microseal &#39;B&#39; PCR Plate Sealing Film, adhesive, optical</td>
			<td>Bio-Rad</td>
			<td>MSB1001</td>
			<td>For real-time PCR plate sealing</td>
		</tr>
		<tr>
			<td>Research plus Single-Channel Pipette, Gray; 0.5-10 &#181;L</td>
			<td>Eppendorf</td>
			<td>UX-24505-02</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>PIPETMAN Classic Pipets, P20</td>
			<td>Gilson</td>
			<td>F123600G</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>PIPETMAN Classic Pipets, P200</td>
			<td>Gilson</td>
			<td>F144565</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Rainin L-1000XLS Pipet-Lite XLS LTS Pipette 100-1000 &#181;L</td>
			<td>Gilson</td>
			<td>17011782</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Glycogen, RNA grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0551</td>
			<td>Improves total RNA isolation efficiency</td>
		</tr>
		<tr>
			<td>Posi-Click 1.7 mL Tubes, natural color</td>
			<td>Denville</td>
			<td>C2170</td>
			<td>RNA isolation and storage; reagent mix</td>
		</tr>
		<tr>
			<td>Thermal Cycling Tubes -0.2 mL Individual Caps, Standard 0.2 mL tubes with optically</td>
			<td>Denville</td>
			<td>C18098-4 (1000910)</td>
			<td>Reverse transcription reaction</td>
		</tr>
		<tr>
			<td>Sharp 10 Precision barrier Tips</td>
			<td>Denville</td>
			<td>P1096-FR</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Sharp 20 Precision barrier Tips</td>
			<td>Denville</td>
			<td>P1121</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Sharp 200 Precision barrier Tips</td>
			<td>Denville</td>
			<td>P1122</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Tips LTS 1 mL Filter</td>
			<td>Rainin</td>
			<td>RT-L1000F</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>miScript Primer Assay (200)</td>
			<td>Qiagen</td>
			<td>(it changes according to the miRNA)</td>
			<td>For real-time PCR analysis</td>
		</tr>
		<tr>
			<td>Gradient Master ver 5.3 Model 108</td>
			<td>BioComp Instruments</td>
			<td></td>
			<td>For preparation of sucrose gradients</td>
		</tr>
		<tr>
			<td>trichloroacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>T6399</td>
			<td></td>
		</tr>
		<tr>
			<td>acetone</td>
			<td>Sigma Aldrich</td>
			<td>650501</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris hydrochloride</td>
			<td>Amresco</td>
			<td>M108</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol</td>
			<td>Fisher Scientific</td>
			<td>BP172</td>
			<td></td>
		</tr>
		<tr>
			<td>iodoacetamide</td>
			<td>Gbiosciences</td>
			<td>RC-150</td>
			<td></td>
		</tr>
		<tr>
			<td>sequencing grade modified trypsine, porcine</td>
			<td>Promega</td>
			<td>V5111</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium bicarbonate</td>
			<td>BDH</td>
			<td>BDH9206</td>
			<td></td>
		</tr>
		<tr>
			<td>formic acid, Optima LC/MS</td>
			<td>Fisher Chemical</td>
			<td>A117</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol, Optima LC/MS</td>
			<td>Fisher Chemical</td>
			<td>A454</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile, Optima LC/MS</td>
			<td>Fisher Chemical</td>
			<td>A996</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind tubes 0.5 mL</td>
			<td>Eppendorf AG</td>
			<td>22431064</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind tubes 1.5 mL</td>
			<td>Eppendorf AG</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>HLB &#181;Elution plate 30 &#181;m</td>
			<td>Oasis</td>
			<td>186001828BA</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac concentrator</td>
			<td>Thermo Scientific</td>
			<td>Savant SPD2010</td>
			<td></td>
		</tr>
		<tr>
			<td>sonicator</td>
			<td>Qsonica</td>
			<td>Oasis180</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Thermo Scientific</td>
			<td colspan="2">Sorvall Legend micro 21R</td>
		</tr>
		<tr>
			<td>LC trap column PepMap 100 C18</td>
			<td>Thermo Scientific</td>
			<td>160454</td>
			<td></td>
		</tr>
		<tr>
			<td>LC separation column PepMap RSLC C18</td>
			<td>Thermo Scientific</td>
			<td>164536</td>
			<td></td>
		</tr>
		<tr>
			<td>mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td colspan="2">Orbitrap Elite ion trap mass spectrometer</td>
		</tr>
		<tr>
			<td>MSConvert software</td>
			<td>ProteoWizard Toolkit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorcerer-SEQUEST software</td>
			<td>Sage-N Research, Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dynamic proteomic and mirna analysis of polysomes from isolated mouse heart after langendorff perfusion

단백질 번역에 동적 변화에 연구 전문된 방법 필요합니다. 여기 우리는 국 소 빈 혈과 reperfusion polysome 프로 파일링과 함께 끼얹는다 절연된 마우스 마음을 사용 하 여 응답에서 새로 합성 된 단백질에 변화를 검사 합니다. 단백질 번역의 동적 변화를 더 이해 하기 우리 mRNAs cytosolic 리보솜 (polyribosomes 또는 polysomes)와 함께 로드 된 또한 미토 콘 드리 아 polysomes 복구 하 고 비교에서 mRNA와 단백질 분포 특징에 높은-효율성 분수 (수많은 리보솜 mRNA에 부착), 낮은-효율성 (적은 리보솜 부착) 또한 미토 콘 드리 아 polysomes, 그리고 번역 비 분수를 포함 하는. miRNAs는 번역 되 고, 번역의 효율성을 줄일 mRNAs와 연결할 수도, 우리는 분수에 걸쳐 miRNAs의 분포를 조사. MRNAs, miRNAs, 및 단백질의 분포는 글로벌 아니 흐름 허 혈과 reperfusion의 30 분 후 30 분 후에 기저 끼얹는다 조건 하에서 시험 되었다. 여기에 우리가이 분석을 수행 하는 데 사용 하는 방법을 제시-특히,이 자당 기온 변화도에서 단백질 추출의 최적화에 대 한 접근은 하지 설명 하기 전에-몇 가지 대표적인 결과 제공.

mRNA 분석 mRNA 결과 각 분수 (그림 3A); 특정 mRNA의 배포로 표현 될 수 있습니다. 정량화, polyribosomal 번역 분수를 결합 하 고 nontranslating 분수 번역 mRNA 풍부의 비율을 제시 번역 비 분수 (그림 3B), 비교. 추가 정보는 별도로에서 낮은 효율 polysome 분수 (그리고 nontranslating 분수에서 별도) 고효율 polysome 분수를 검사 하 여 얻었?...

Watch this Scientific Journal Video about Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion at JoVE.com

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

여기 선물이 polysome 끼얹는다 절연된 마우스 마음에 프로 파일링을 수행 하는 프로토콜. 우리 심장 관류, 프로 파일링, polysome polysome 분수 mRNAs, miRNAs, 및 polysome 프로테옴의 분석에 대 한 방법을 설명 합니다.

동적 Proteomic 그리고 미르 Langendorff 관류 후 절연 마우스 마음에서에서 Polysomes의 분석

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

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Biochemistry

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

블루 기본 페이지와 단백질 상관 관계 프로파일 링에 의한 선호도 정제 단백질 복합체 해결

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

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생화학

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

Proteomic 분석에 대 한 인간의 망막과 맥락 막의 RPE의 해 부

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

Hepatocytes에서 마우스 간 사인 내 피 세포의 정화 관류

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

Split-BioID ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

그들의 네이티브 셀룰러 환경에서 상황에 맞는 단백질 복합물의 분할-BioID-Proteomic 분석

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

동적 단백질 복합물 분석에 조립 하 고 질량 분석 및 전자 현미경을 사용 하 여 Biolayer 간섭계 바이오 센서에서 발표

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

생체 외에서 교정 무료 단백질 호모-oligomerization 상업 계측 및 무료, 오픈 소스 밝기 분석 소프트웨어를 사용 하 여의 정량화

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

낮은 산소 환경에서 인간 대 식 세포 분극의 Proteomic 분석

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

유비퀴틴 및 유비퀴틴과 같은 종속 적 번역 후 수정 및 중대한 변경 식별 프로파일링

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Proteomic Profile of EPS-Urine through FASP Digestion and Data-Independent Analysis

FASP 소화 및 데이터 독립적 분석을 통한 EPS-소변의 프로테오믹 프로파일

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F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

FÃƒÆ’Ã‚Â¶rster Resonance Energy Transfer Measurements in Living Plant Cells

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Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...