This work was supported by a grant from the Canadian Institutes for Health Research (MOP-86514).

쥐 실험 절차는 맥길 대학 (프로토콜 4966)의 동물 관리위원회에 의해 승인되었다. 배양 된 세포에서 단백질 추출물 1. 준비 <ol><li> 얼음처럼 차가운 인산 완충 생리 식염수 10 ㎖ (PBS)로 두 번 배양 세포를 씻으십시오. </li><li> 고무 경찰관 또는 1.5 ml의 microcentrifuge 관에 얼음처럼 차가운 PBS, 전송 현탁액 1 ㎖의 플라스틱 셀 스크레이퍼 중 하나에 부착 된 세포를 다 쳤어요. </li><li> 4 ° C에서 700 XG에 5 분의 microcentrifuge에 스핀. 대기음 PBS. </li><li> 10 7 세포 당 얼음처럼 차가운 세포질 용해 버퍼 100 ㎕ (표 1)를 추가하고 아래로 피펫. </li><li> 얼음에서 20 분 동안 인큐베이션. </li><li> 4 ° C에서 마이크로 원심에서 최고 속도로 10 분 동안 스핀. </li><li> 취소 펠렛. 새로운 1.5 ml의 microcentrifuge 관에 뜨는을 전송하고 얼음에 보관하십시오. </li><li> DET브래드 포드 분석 (23)를 사용하여 - (10 ㎍ / μL 일반적으로 1) 순백의 단백질 농도. </li><li> 사용할 때까지 -80 ° C에서 나누어지는 및 저장 세포 추출합니다. </li></ol> 마우스 간 및 비장에서 단백질 추출물 2. 준비 <ol><li> CO 2 흡입으로 마우스를 안락사. </li><li> 해부 보드 위에 깨끗한 패드의 안락사 동물을 놓습니다. 가위로 복부를 엽니 다. </li><li> 가위 집게를 사용하여 간 및 비장을 해부하고, 약 50 ml의 빙냉 PBS에서 각 조직을 헹군다. </li><li> 즉시 메스 작은 조각으로 조직을 절단 (예 : 약 1-2mm 3). </li><li> 지체없이 신선한 cryotube에 조직의 조각을 넣고 액체 질소를 고정 스냅. 스토어 스냅인 냉동 조직 씩을 사용할 때까지 -80 ° C에서. </li><li> 0.5 ㎖의 - 0.25 - (2 내지 3mm 정도 1) 동결 조직의 한 조각 균질화세포질 빙냉 용해 완충액 (표 1) 10 초 동안 조직 균질화. </li><li> 20 분 동안 얼음에 1.5 ml의 microcentrifuge 관과 진정으로 균질을 전송합니다. </li><li> 4 ° C에서 마이크로 원심에서 최고 속도로 10 분 동안 스핀. </li><li> 새로운 1.5 ml의 microcentrifuge 관에 펠렛 및 전송 상층 액을 제거한다. 얼음에 보관하십시오. </li><li> 브래드 포드 분석 (23)를 사용하여 - (10 ㎍ / μL 일반적으로 1) 단백질 농도를 결정합니다. </li><li> 사용할 때까지 -80 ° C에서 나누어지는 및 저장 세포 추출합니다. </li></ol> 방사성 표지 IRE 프로브 3. 준비 <ol><li> 제한 효소 XbaI으로 (플라스미드 당 1 μg의 U), IRE 서열의 하류로 절단하여, 1 시간 동안 37 ° C에서 배양하여 IRE 함유 플라스미드 I-12.CAT 22 선형화. 선형화 된 플라스미드는 시험 관내 전사를위한 템플릿으로 사용됩니다. </li><li> 설정N 체외 20㎕의 전체 부피에서 전사 반응. 표 2에 나타낸 스톡 솔루션을 사용하고 추가 1 μL 선형화 된 플라스미드 주형 4 μL 전사 버퍼; ATP / CTP / GTP 믹스 1 ㎕를 혼합, 10 ㎕의 [α- 32 P] -UTP, 2 μL 디티 오 트레이 톨, 1 ㎕의 RNA 분해 효소 억제제 및 1 μL의 T7 RNA 폴리머 라제. 로 pipetting 아래로 섞는다. </li><li> 1 시간 24 40 ° C에서 품어. </li></ol> 방사성 표지 IRE 프로브 4. 정제 <ol><li> 피펫 팅 아래로 0.5 M EDTA, pH가 8 믹스 1 μl를 추가하여 시험 관내 전사 반응을 종료합니다. </li><li> 더 나은 침전 캐리어로, 10 ㎎ / ㎖의 tRNA의 10 μl를 추가합니다. 로 pipetting 아래로 섞는다. </li><li> 3 M 암모늄 아세테이트 82.5 μl를 추가합니다. 와동에 의해 섞는다. </li><li> 에탄올 273 μl를 추가합니다. 와동에 의해 섞는다. </li><li> 5 분 동안 RT에 서 보자. </li><li> RT에서 마이크로 원심에서 최고 속도로 10 분 동안 스핀. 취소 상층 액. </li><li> 워시는 70 % 에탄올 100 ㎕로 펠렛. </li><li> RT에서 마이크로 원심에서 최고 속도로 10 분 동안 스핀. 취소 상층 액. </li><li> 10 분 동안 공기 건조 펠렛. </li><li> 이중 증류수 100 ㎕에 재현 탁 펠릿은, 이전에 H 2 O를 멸균 </li><li> 사용할 때까지 -80 ° C에서 액체 섬광 계수기의 방사능, 나누어지는 방사성 동위 원소 표지의 IRE 프로브와 저장소를 정량화. 냉동 분취 최대 3 주 동안 사용될 수있다. </li></ol> EMSA에 대한 기본 폴리 아크릴 아미드 겔 5. 준비 <ol><li> 1.5 mm 스페이서와 빗을 사용하여 겔 (16 × 16cm)를 조립합니다. </li><li> 표 3에 도시 된 원액을 사용하여, 6 % 폴리 아크릴 아미드 겔 네이티브을 제조 7.5 ㎖의 40 % 아크릴 아미드의 혼합 :. 비스 아크릴 아미드, 5X TBE 5 ㎖과 37.5 ml에더블 증류수 H 2 O </li><li> 10 % 새롭게 제조 된 황산 암모늄 (APS) 25 μL의 테트라 메틸 에틸렌 디아민 (TEMED)의 0.5 ML을 추가합니다. </li><li> 즉시 젤에 아크릴 아미드 솔루션을 붓고 중합 할 수 있습니다. 약 30 분 동안 기다립니다. </li><li> 상기 겔 전기 영동 장치를 조립하여 0.5X TBE의 탱크를 채우기와 전원 연결. </li></ol> 6. 전기 이동 기동성 교대 분석 실험 <ol><li> 10 μL의 총 부피에서 세포질 또는 세포 용해 완충액 (표 1)과 조직으로부터의 단백질 추출물의 25 μg의 희석 (저급 단백질 농도도 사용될 수있다). 얼음에 보관하십시오. 25 : 4 휴면 IRP1 (2 % 최종 농도)를 활성화 2- 머 캅토 에탄올 (2-ME)로 희석 : 필요한 경우, 1의 1 μl를 추가합니다. </li><li> 95 °에서 두 번 증류수에 방사성 동위 원소 표지 IRE 프로브를 희석 2 O 200,000 CPM / μL, 열 변성에1 분 C, 그리고 적어도 5 분 동안 실온에서 냉각. </li><li> 단백질 추출물에 방사성 동위 원소 표지 IRE 프로브의 1 μl를 추가하여 EMSA 반응을 설정합니다. </li><li> 실온에서 20 분 동안 품어. </li><li> 반응 50 ㎎ / ㎖ 헤파린의 1 μl를 추가 (프로브 (9)와 비 특정 단백질 상호 작용을 억제하기 위해) 또 다른 10 분 동안 배양을 계속합니다. -80 ° C에서 스톡 헤파린 용액 (50 ㎎ / ml) 및 저장소 나누어지는. </li><li> 긴 방사성 표지 된 프로브 (&gt; 60 개 뉴클레오티드)를 사용하는 경우, 1 μL의 RNase T1 (1 U / μL)를 첨가하고, RT에서 10 분 동안 인큐베이션 프로브 비특이적 결합 단백질을 줄이기 위해, 및 RNA / 단백질의 잘 구분할 수 있도록 전기 영동시 복잡한. </li><li> 로딩 버퍼 (80 % 글리세롤 + 브로 모 페놀 블루)의 3 μl를 추가, 혼합 6 % 비 변성 폴리 아크릴 아미드 겔에로드. </li><li> 130 V (5 V / cm)에서 60 분 동안 젤을 실행합니다. </li><li> 티를 ransfer 큰 필터 종이 건조 위에 젤. </li><li> 필름에 노출 및 오토 라디오를 개발한다. 노출 시간은 1 시간 (이하)에서 O / N까지의 범위 일 수있다. </li></ol>

<ol>
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B., Klausner, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation-reduction+and+the+molecular+mechanism+of+a+regulated+RNA-protein+interaction.">Oxidation-reduction and the molecular mechanism of a regulated RNA-protein interaction.</a> Science. 244, 357-359 (1989).</li><li>Meyron-Holtz, E. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+ablations+of+iron+regulatory+proteins+1+and+2+reveal+why+iron+regulatory+protein+2+dominates+iron+homeostasis.">Genetic ablations of iron regulatory proteins 1 and 2 reveal why iron regulatory protein 2 dominates iron homeostasis.</a> EMBO J. 23, 386-395 (2004).</li><li>Huang, F. W., Pinkus, J. L., Pinkus, G. S., Fleming, M. D., Andrews, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+juvenile+hemochromatosis.">A mouse model of juvenile hemochromatosis.</a> J. Clin. Invest. 115, 2187-2191 (2005).</li><li>Sebastiani, G., Pantopoulos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorders+associated+with+systemic+or+local+iron+overload:+from+pathophysiology+to+clinical+practice.">Disorders associated with systemic or local iron overload: from pathophysiology to clinical practice.</a> Metallomics. 3, 971-986 (2011).</li><li>Galy, B., Ferring, D., Hentze, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+conditional+alleles+of+the+murine+iron+regulatory+protein+(IRP)-1+and+-2+genes.">Generation of conditional alleles of the murine iron regulatory protein (IRP)-1 and -2 genes.</a> Genesis. 43, 181-188 (2005).</li><li>Gkouvatsos, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron-dependent+regulation+of+hepcidin+in+Hjv-/-+mice:+Evidence+that+hemojuvelin+is+dispensable+for+sensing+body+iron+levels.">Iron-dependent regulation of hepcidin in Hjv-/- mice: Evidence that hemojuvelin is dispensable for sensing body iron levels.</a> PLoS ONE. 9, 85530 (2014).</li><li>Goforth, J. B., Anderson, S. A., Nizzi, C. P., Eisenstein, R. 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The authors declare that they have no competing financial interests.

여기서, 우리는 IRP1과 IRP2의 IRE 결합 활동을 연구하기 위해 개발 된 프로토콜을 설명하고 우리는 대표적인 데이터를 보여줍니다. 다른 프로브를 사용함으로써,이 프로토콜은 다른 RNA 결합 단백질의 연구를 위해 조정될 수있다. 중요한 단계는 탐침의 크기이다. 정확한 결합 부위를 알 수없는 경우 일반적인 긴 프로브의 사용은 무료 RNA 다르게 마이그레이션되지 않습니다 RNA / 단백질 복합체가 발생...

EMSA는 원래 타겟 DNA 서열과 1,2- DNA 결합 단백질의 관계를 연구하기 위해 개발되었다. 원리는이 문서의 초점이다 RNA / 단백질 상호 작용 3, 비슷합니다. 간단히, RNA는 음전하 폴리 아크릴 아미드 (또는 아가로 오스) 겔에서 전기 영동 비 변성 중에 애노드쪽으로 이동한다. 겔 내에서 마이그레이션 충전량에 비례하는 RNA의 크기에 따라 달라집니다. 특정 RNA에 단백질의 결합은 무료 RNA에 비해 복잡한 마이그레이션하는 느린 이동성을 변경합니다. 이는 분자량의 증가뿐만 아니라, 충전 및 가능한 입체 구조의 변화에​​ 주로 기인한다. 프로브로 표지 된 RNA를 활용하여 &quot;젤 지연&quot;또는 &quot;bandshift&quot;쉽게 모니터링 할 수 있습니다. 32 P - 라벨 RNA 프로브의 사용은 매우 일반적이며 높은 감도를 제공합니다. RNA / 단백질 복합체 무료 RNA의 이동이 감지된다오토 라디오로. 단점 32 P (14.29 일), 방사선 분해로 인해 프로브의 품질의 점진적인 열화의 짧은 반감기이다 방사능 라이센스 방사능 작업 인프라 및 잠재적 생물 안전성 문제의 요구. 따라서, RNA 프로브 레이블을 대체 비 동위 원소 방법은 형광 또는 화학 발광 영상 4,5의 검출을 가능하게 형광 물질이나 바이오틴,와 예를 들어, 개발되었다. 이러한 방법의 제한은 높은 비용과 동위 원소 라벨에 비해 자주 감소 감도 및 RNA / 단백질 상호 작용을 방해하는 비 동위 원소 라벨의 가능성이 있습니다. 비 변성 폴리 아크릴 아마이드 겔은 가장 EMSA 애플리케이션에 적합하며 일반적으로 사용된다. 경우에 따라, 아가 로스 젤 큰 단지의 분석을위한 대안을 제기 할 수 있습니다. EMSA의 주요 장점은 단순성, 감도 및 안정성을 겸비한 4 점이다 &lt;/ SUP&gt;. 분석은 몇 시간 내에 완료 될 수 있고, 정교한 장비를 필요로하지 않는다. RNA / 단백질 상호 작용은 0.1 nm 이하의 낮은 농도에서 EMSA에 의해 검출 될 수 있고, 광범위한 결합 조건의 범위 내에서 (pH가 4.0-9.5, 가의 염 농도 1-300 ㎜, 온도 0-60 ℃). RNA / 단백질 복합체 형성은 필터 결합 분석에 의해 연구 할 수있다. 자유 RNA 프로브가 6을 통과하면서는 니트로 셀룰로오스 필터 RNA / 단백질 복합체의 유지에 기초하여, 간단하고, 빠르고, 저렴한 절차이다. EMSA에 비해, 그것은 RNA 프로브가 다중 결합 부위를 포함하거나 조 추출물이 동일한 사이트에서 프로브에 결합하는 하나 이상의 RNA 결합 단백질이 들어 가지 경우에 한정된다. 여러 RNA / 단백질 상호 작용은 필터 결합 분석에 의해 탐지되지 것이지만, 그들은 용이 EMSA에 의해 시각화 될 수있다. 어떤 경우에는, 시각화 이브이다N 가능한 경우, 겔에 더 지체를 산출, EMSA 반응에 RNA 결합 단백질 중 하나에 대한 항체를 추가하여 공동 마이그레이션 (예를 들어, 인간의 IRP1 / IRE 및 IRP2 / IRE 단지) 두 RNA / 단백질 복합체 ( &quot;supershift&quot;) 7. EMSA 널리 철 대사 8-10의 전사 후 레귤레이터이다 IRP1과 IRP2을 연구하는 데 사용되었다. 그들은 몇 가지의 mRNA (11)의 UTRs 내에서 IRES, 계통 발생 학적으로 보존 헤어핀 구조에 결합함으로써 작동합니다. IRES 먼저 페리틴 (12)와 트랜스페린 수용체 1 (TfR1) (13), 각각 철 저장 및 흡수의 단백질을 암호화하는 mRNA를 발견했다. 나중에, IRES는 적혈구 특정 aminolevulinate 신타 (ALAS2) 14 미토콘드리아 aconitase 15 ferroportin 16, 2가 금속 수송 체 1 (DMT1) 17, 저산소증 유도 성 인자 (2)에서 발견 된 <em&gt; α (HIF2α) (18), 및 기타의 mRNA 19-21. UTR &#39;TfR1의 mRNA는 3에서 여러 IRES를 포함하는 동안, UTR&#39;프로토 타입 H-와 L-페리틴의 mRNA는 5 일 IRE가 포함되어 있습니다. IRE / IRP의 상호 작용은 특히 입체 43S 리보솜 서브 유닛의의 연결을 차단하여 페리틴 mRNA의 번역을 억제; 또한, 그들은 endonucleolytic 분열에 대한 TfR1 mRNA의 안정화. IRP1과 IRP2 공유 광범위한 서열의 유사성과 철에 굶주린 세포에서 높은 IRE 결합 활성을 나타낸다. IRP2은 프로 테오 열화를 겪는 동안 철 구비 세포에서, IRP1는 그것 IRE 결합능 희생 aconitase 세포질로 변환 쿠반의 Fe-S 클러스터를 조립한다. 따라서, IRE / IRP 상호 작용은 세포 철 상태에 의존 할뿐만 아니라, H 2 O 2, 산화 질소 (NO) 또는 저산소증과 같은 다른 신호들에 의해 조절된다. 여기서는 E에 의해 조 세포 및 조직 추출물에서 IRE - 결합 활성을 평가하기위한 프로토콜을 묘사MSA. 우리는 IRE 서열은 원래 의해 하류 T7 RNA 중합 효소 사이트의 센스 방향에 도입 된 플라스미드 DNA 템플릿 (I-12.CAT)로부터 시험 관내 전사에 의해 생성 된 32 P - 표지 H-페리틴 IRE 프로브를 사용 어닐링 합성 올리고 뉴클레오티드 (22)의 복제.

<table border="0" cellpadding="0" cellspacing="0" width="430">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:195px;">Name of the Materials&#160;</td>
			<td style="width:119px;">Company</td>
			<td style="width:117px;">Catalog Number&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">leupeptin</td>
			<td>SIGMA</td>
			<td>L2884</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PMSF</td>
			<td>SIGMA</td>
			<td align="right">78830</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BioRad Protein Assay</td>
			<td>BIORAD</td>
			<td>500-0006</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">T7 RNA polymerase</td>
			<td>Thermoscientific</td>
			<td>EPO111</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rnase Inhibitor</td>
			<td>Invitrogen</td>
			<td>15518-012</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">UTP [alpha-32P]</td>
			<td>Perkin-Elmer</td>
			<td>NEG507H</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scintillation liquid</td>
			<td>Beckman Coulter</td>
			<td align="right">141349</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">heparin</td>
			<td>SIGMA</td>
			<td>H0777</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rnase T1</td>
			<td>Thermoscientific</td>
			<td>EN0541</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Name of the Equipments&#160;</td>
			<td>Company</td>
			<td>Catalog Number&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue Ruptor</td>
			<td>Qiagen</td>
			<td align="right">9001271</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scintillation counter</td>
			<td>Beckman Coulter</td>
			<td>LS6500</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Protean II xi Cell&#160;&#160;</td>
			<td>BIORAD</td>
			<td>165-1834</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">20 wells combs 1.5mm thick</td>
			<td>BIORAD</td>
			<td>165-1868</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1.5 mm spacers</td>
			<td>BIORAD</td>
			<td>165-1849</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PowerPac</td>
			<td>BIORAD</td>
			<td>164-5070</td>
		</tr>
	</tbody>
</table>

electrophoretic mobility shift assay (emsa) for the study of rna-protein interactions: the ire/irp example

RNA / 단백질 상호 작용은 전사 후 조절 경로에 대한 중요합니다. 가장 특징 세포질 RNA 결합 단백질 중 철 조절 단백질, IRP1과 IRP2입니다. 그들은하여 mRNA를 번역 또는 안정성을 제어하는​​ 여러 대상의 mRNA의 번역되지 않은 지역 (UTRs) 내에서 반응 요소 (IRES)을 다림질 결합한다. IRE / IRP 상호 작용은 널리 EMSA에 의해 연구되고있다. 여기서, 우리는 다른 RNA 결합 단백질의 활성을 평가하기 위해 일반화 될 수 IRP1과 IRP2 IRE의 결합 활성을 EMSA 분석 프로토콜을 설명한다. RNA 결합 단백질, 또는이 단백질의 정제 된 제제를 함유하는 조단백질 해물, 복합체 형성을 가능하게 표지 된 32 P-RNA 프로브의 과량과 함께 배양된다. 헤파린 결합 프로브 비 특정 단백질을 배제하기 위해 추가됩니다. 이어서, 혼합물을 폴리 아크릴 아미드 겔상에서 비 변성 전기 영동에 의해 분석된다. 무료 프로브, 빠른 마이그레이션하는 동안 RNA / 단백질 복합체 전시 지체 이동; 그러므로, 절차는 또한 &quot;겔 위상차&quot;또는 &quot;bandshift&quot;분석 불린다. 전기 영동 완료 후, 겔을 건조 및 RNA / 단백질 복합체,뿐만 아니라 자유 프로브는, 오토 래디오 그래피에 의해 검출된다. 프로토콜의 전반적인 목적은 감지 IRE / IRP 및 기타 RNA / 단백질 상호 작용을 정량화하는 것이다. 또한, EMSA는 조사중인 RNA / 단백질 상호 작용의 특이성 결합 친화력 및 화학량 론을 결정하는 데 사용될 수있다.

섹션 3 및 4 프로토콜에 기재된 방사성 표지 된 프로브 IRE 제조 하였다. 프로브의 서열은 5&#39;-CUG GGGCGAAUUC GAGCUCGGUA CCCGGGGAUC UUCAA C C AGUGC UUGGA CGGAUCCU-3 &#39;이었다; 굵은 뉴클레오티드는 중요한 IRE 기능입니다 짝이없는 C 잔류 루프를 나타냅니다. 프로브의 특정 방사능 4.5 × 109 CPM / μg의 RNA의이었다. 활성 IRE는 결합에 철 교란의 효과를 평가하기 위해, 뮤린 RAW...

Watch this Scientific Journal Video about Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example at JoVE.com

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

여기에서 우리는 RNA / 단백질 상호 작용을 분석하는 프로토콜을 제시한다. 전기 영동 이동성 변화 분석 (EMSA)이 네이티브 겔 전기 영동시 RNA / 단백질 복합체 및 무료 RNA의 차분에 기초하여 이동된다. 방사성 표지 된 RNA 프로브를 사용함으로써, RNA / 단백질 복합체는 오토 라디오 그래피에 의해 시각화 될 수있다.

IRE / IRP 예 : RNA - 단백질 상호 작용의 연구에 대한 전기 이동 기동성 교대 분석 실험 (EMSA)

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

electrophoretic-mobility-shift-assay-emsa-for-study-rna-protein

Research

JoVE Journal

Biology

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

53.2K 조회수.  Jewish General Hospital.  여기에서 우리는 RNA / 단백질 상호 작용을 분석하는 프로토콜을 제시한다. 전기 영동 이동성 변화 분석 (EMSA)이 네이티브 겔 전기 영동시 RNA / 단백질 복합체 및 무료 RNA의 차분에 기초하여 이동된다. 방사성 표지 된 RNA 프로브를 사용함으로써, RNA / 단백질 복합체는 오토 라디오 그래피에 의해 시각화 될 수있다. 

비디오: Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

연구

생물학

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Angiogenesis 연구에 최적화된 섬유소 젤 비드 분석

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

컴퓨터가 만든 동물 모델 자극

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Cryopreserved 샘플 복구를 위해 손수 함 장치 실수로 극저온 저장 탱크 떨어졌고

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

단백질 - 단백질 상호 작용 연구를 자성 비즈를 사용하여 액상 동질 캡처 분석 : Poliovirus-Nanobody의 예

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

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 ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

함수의 연구, 상호 작용, 그리고 번역 후 수정을위한 신진 효모 단백질 추출 및 정제

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

단백질-DNA 상호 작용의 연구에 대한 단일 분자 조작 및 이미지를 결합

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

애기 장대 단백질 DNA 상호 작용의 식별을위한 염색질 면역 침전 분석<em&gt; 생체</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

전기 영동 이동성 분석 (EMSA) 및 Shift 키를 사용하여 기능 비 코딩 유전자 변형에 대한 심사 DNA 화성 강수 분석 (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...