이 작품은 국립 과학 재단 (National Science Foundation) 부여 IOS-0817658에 의해 지원되었다.

이 프로토콜의 제브라 피쉬 절차는 버지니아 커먼 웰스 대학에서 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었습니다. 시약 1. 준비 <ol><li> 4 % PFA / PBS. 흄 후드에서 파라 포름 알데히드의 8g (PFA)를 달아. 여전히 흄 후드에서, ~ 80 ml를 50 ° C로 교반 및 가열 증류수 H 2 O를 건조 PFA를 용해하는 동안. PFA 완전히 용해되어 용액이 명확 할 때까지 신선한 1N NaOH를 10 방울 - 교반하면서, 3을 추가합니다. 열에서 제거하고 100 ml의 2 배 인산염 완충 식염수 (PBS)를 추가합니다. 증류수 H 2 O 200 ml의 부피를 가져와 실온으로 냉각하고 사용하기 전에 7.4의 산도를 확인합니다. 4 ° C에서 어둠에 보관하지만 1 주일 이내에 사용한다. </li><li> 어떤 보충없이 100 % 메탄올을 사용합니다. 어떤 보충없이 95 % 에탄올을 사용합니다. </li><li> 인산염 완충 트윈 (PBT)를 준비합니다. 0.1 % 트윈 -20으로 인산 완충 식염수 (PBS)를 보완. 0을 추가합니다.1X PBS 100 ㎖에 20 % 트윈 -20 스톡 용액 5 ㎖. 실온에서 보관하십시오. </li><li> 인산염 완충 트리톤-X (PBTx)를 준비합니다. 0.1 % 트리톤 X-100으로 PBS를 보충합니다. PBS 100 ㎖에 20 % 트리톤 X-100 주식의 0.5 ML을 추가합니다. 실온에서 보관하십시오. </li><li> 10 % NGS / PBTx를 준비합니다. 정상 염소 혈청 (NGS) 블록은 비 - 특이 적 결합 부위. -20 ° C에서 분취에 보관 NGS. 사용 9 ml의 PBTx 1 ml에 추가합니다. </li><li> 50 % 글리세롤 / PBS를 준비합니다. 배 PBS 100 % 글리세롤의 완전히 동일한 부분을 섞는다. 실온에서 보관하십시오. </li></ol> 2. 배아 고정 <ol><li> . (. 예를 들어, β - 굴지 : CAAX-GFP) 사육 구하는 야생형 (AB 및 WIK) 또는 형질 전환 자연 교배를 통해 배아. 원하는 경우 설명 된대로, 구조 또는 morpholinos와 배아를 주입. (15, 16) </li><li> . 배아 증액 배아 모아서 바와 같이 염색을 차단하기 위해 0.003 %, 1- 페닐 -2- 티오 우레아 (PTU)의 존재하에 28.5 ℃에서 배양한다. 17 </su<html> P&gt; </li><li> 배아가 원하는 발달 단계, 18에 도달하면. 수정, &#160; , MESAB를 이용하여 배아 마취 최대한 시스템의 물을 제거하고 신선한 3 4 % PFA / PBS를 추가 - RT에서 4 시간. 참고 : 20 시간 게시물 수정 (HPF)보다 적은 시간에, dechorionation하기 전에 수정합니다. HPF (20) 이후에 때때로, 해부 현미경 하에서 미세 점 두 쌍의 집게를 이용하여 고정하기 전에 dechorionate. </li><li> 솔루션을 변경. 넓은 구멍 피펫을 사용하여 배아로 이동 한 바와 같이.의 microcentrifuge 튜브 덮인 1.5 ml의 17 변경 솔루션, 단순히 배아가 원심 분리하지 않고 중력에 의해 정착 할 수 있도록하여. </li><li> 후 정착액 3 후 -. 4 시간, 최소 48 시간 동안 -20 ° C에서 100 % 메탄올 저장 교체, PFA / PBS를 제거합니다. 참고 : 최대 P-CaMK-II 면역를 들어, 일주일 총 메탄올 저장 시간을 제한 할 수 있습니다. </li></ol>&quot;&gt; 3. 면역 염색 항목의 태아 <ol><li> 출장 1.5 ML의 microcentrifuge 관에서 각 실험 조건 10 배아의 최소 배치하고 레이블을 붙입니다. </li><li> 배아가 정착하도록 허용합니다. 메탄올을 제거하고 폐기하고 다음 단계에서 나타낸 바와 같이, 에탄올의 농도를 감소 0.5ml를 함유 프로그레시브 세척하여 재수. 각 단계는 락과 5 분 세척입니다. </li><li> 점진적이 5 솔루션 재수 : 66 % 에탄올 / 33 % PBTx, 33 % 에탄올 / PBTx은 100 % PBTx 100 % PBTx (이 PBTx 첫 반복이다), 100 % PBTx (이 두 번째 반복 인 66 % PBTx). </li><li> 마지막 PBTx 세척을 제거합니다. 0.5 ml의 10 % NGS / PBTx를 추가합니다. 부드러운 락과 실온에서 적어도 1 시간 동안 품어 후 제거 솔루션을 폐기합니다. </li><li> 10 % NGS / PBTx에 희석 차 항체 솔루션을 준비합니다. 공동 면역 염색 경우, 먼저 높은 친 화성 항체를 사용합니다. 500 : 본 연구를 위해, 1 희석 마우스 항 아세틸 튜 불린 단클론 항체로 배양한다. exampl에 대한즉, 10 개의 샘플이 10 % NGS / PBTx 3 ml의 스톡 항체 용액 6 ㎕를 결합하고,이 각 튜브에 0.3 ㎖에 분산이있는 경우. </li><li> 모든 배아를 체험하기 위해 각 튜브에 희석 차 항체 0.5 ml의 - 0.2을 추가합니다. 실온에서 부드럽게 흔들 O / N로 품어. </li><li> 아침에 제거하고 항체 용액을 버린다. 초과 일차 항체를 씻어 0.5 ml의 2 % NGS / PBTx를 추가합니다. 부드럽게 5 분 동안 바위. 제거 및 솔루션을 폐기합니다. 두 번 반복합니다. </li><li> 머리 위의 조명을 어둡게하고 각 조건이 적어도 0.5 ml에 포함되도록 10 % NGS / PBTx에서 적절한 형광 접합 된 이차 항체를 희석. (500) 희석 : 마우스 항 - 아세틸 화 튜 불린 차 항체로, 1에서 녹색 형광 염료 염소 항 - 마우스 IgG를 사용합니다. </li><li> 부드러운 어둠 속에서 RT에서 흔들 4 시간 동안 차 항체를 품어. 희미하게 불빛 아래 어두운 용기에 배양 또는 알루미늄 호일에 포장하여 샘플 프로세스, 지금부터. </li><li>인큐베이션의 끝에, 제거 차 항체 용액을 버린다. 0.5 ml의 2 % NGS / PBTx 세탁에 추가. 부드럽게 5 분 동안 바위. 제거 및 솔루션을 폐기합니다. 두 번 반복합니다. </li><li> 공동 면역 경우 제 차 항체보다는 다른 종으로부터 제 차 항체를 얻었다. 이 연구에서 붉은 형광 염료 결합 염소 항 - 토끼 IgG를 이차 항체에 의해 토끼 항 - 포스 CaMK-II 항체를 따릅니다. </li><li> 토끼 항 - 포스 CaMK-II 항체 1:20 희석. 배아의 각 튜브를 들어, 다음, 0.3 ml의 10 % NGS / PBTx에 중단 주식 항체 용액 15 μl를 추가합니다. </li><li> 배아가 몰입과 조명이 희미하게되어 있는지 확인합니다. 어둠 속에서 실온에서 부드럽게 흔들 O / N로 품어. </li><li> 희미한 불빛 아래에서 아침에 제거하고 항체 용액을 버린다. 0.5 ml의 2 % NGS / PBTx를 추가합니다. 부드럽게 5 분 동안 바위. 제거 및 솔루션을 폐기합니다. 두 번 반복합니다. </li><li> 희미한 간접 조명을 유지하고 적절한 형광 conju 충분히 희석문이 이차 항체 있도록 각 조건은 적어도 0.5 ml에 포함되어 있습니다. 500 10 % NGS / PBTx : 본 연구에서는 붉은 형광 염료 결합 염소 항 - 토끼 IgG를 이차 항체 (적색 채널) 1을 희석. </li><li> 부드러운 어둠 속에서 RT에서 흔들 4 시간 동안 제 2의 2 차 항체를 품어. </li><li> 이 배양 희미한 조명 아래 끝에서 제거 차 항체 용액을 버린다. 0.5 ml의 PBTx을 추가합니다. 부드럽게 5 분 동안 바위. 제거 및 솔루션을 폐기합니다. 두 번 반복합니다. 쇼핑몰 PBTx 50 % 글리세롤 / PBS 하나로 촬상 절차에 따라. </li></ol> 4. 공 촛점 이미징 및 처리 <ol><li> 마운트 이미징 배아. 유리 슬라이드에 5 배아 - 1을 배치합니다. 메인 커버 슬립 및 챔버의 각각의 측면 상에 커버 슬립 단편을 사용하여 슬라이드 사이에 챔버를 만든다. 일반적으로, 네 # 1 커버 슬립은 밀봉하지 않고 이러한 스페이서 스택을 만드는 데 사용됩니다. 참고 : 설치 매체가 필요하지 않습니다. </li><li> 100X 기름 침지를 사용하여목적은 전송 된 빛을 사용하여 초점 단일 배아를 가지고 있습니다. 빛을 끕니다. 공 초점 현미경을 켭니다. 적절한 레이저 (녹색 및 / 또는 적색) 켜져 있고 원격 포커스 악세사리가 종사을 확인합니다. </li><li> 공 초점 프로그램을 켭니다. 에서 &quot;획득 바,&quot;적절한 목표를 선택합니다. &quot;XY 기본&quot;줄에 1,024 버튼을 클릭하여 1,024에 이미지 크기를 설정합니다. </li><li> &quot;레이저 및 감지기&quot;줄에서 각 채널에 대한 레이저 및 검출기를 켭 빨간색 488 상자와 녹색 568 상자를 클릭합니다. 매체에 핀홀 설정하고 시각화하는 각 채널의 &quot;게인&quot;줄의 게인을 조정합니다. </li><li> &quot;취득 설정&quot;줄에서 이미지를 획득 시작 &quot;라이브&quot;를 클릭합니다. &quot;보기 설정&quot;줄에서 &quot;라이브&quot;창에서 가운데​​로 &quot;일체형 줌을 강제로&quot;상자를 선택 취소합니다. </li><li> &quot;설정 획득&quot;줄에서 &quot;Z&quot;탭에서, 이미지의 레이어를 통해 단계. 뉴를 선택한 후광학 섹션의 mber, 이미지에 통합 Z-평면의 &quot;중심&quot;의 어느 시점으로 이동합니다. </li><li> &quot;Z&quot;탭에서 작은 빨간 &quot;참조&quot;상자를 클릭합니다. 이 &quot;중앙&quot;으로서 선택 시점 RFA 영점 것이다. &quot;스텝 크기&quot;라고 표시된 상자에 (이상적입니다 μm의 μm의 0.25 내지 2.0) 층의 두께를 선택합니다. &quot;파일 크기&quot;상자에 주목하고 1GB로 제한하려고합니다. 주 : 일반적으로 0.5 내지 40 부까지의 광학 - 1.0 μm의 수득되지만, 섹션들의 수는 실험적으로 결정될 수있다. </li><li> 이미지의 상단 극단적 인을 찾으려면 &quot;최고&quot;옆에있는 원을 클릭하고 Z 층을 통해 이동합니다. 이제 다음 &quot;바닥&quot;에 동그라미를 누르면 컴퓨터는 중심으로 층으로 다시 이미지를 취할 것입니다. 다시 이미지의 하단 극단적 인을 찾기 위해 레이어를 통해 이동합니다. </li><li> 레이저를 중지 상자 &quot;취득 설정&quot;에서 다시 &quot;라이브&quot;를 클릭취득. 이 패널에서 빨간 평균 표시 박스와 Z-스택을 클릭합니다. 이 상자는 녹색으로 바뀝니다. </li><li> &quot;취득 설정&quot;상자에서 이미지의 전체 시리즈를 취득하는 &quot;싱글&quot;을 클릭합니다. 이 두 채널에서 전체 Z - 스택을 통해 검색과 같이 진행 상황을 모니터링 할 수 있습니다. </li><li> 저장 볼륨 창을 클릭 클릭 &quot;다른 이름으로 저장&quot;및 파일 이름을 지정합니다. &quot;.ids&quot;파일로 저장합니다. 볼륨 Z-스택이 열려있는 동안, 파일을 렌더링 데이터 풀다운 메뉴에서 &quot;렌더링 볼륨&quot;을 클릭 선택합니다. TIFF 파일로 렌더링 된 파일을 저장합니다. 현재 문서는 인쇄물에 표시됩니다 투사 된 이미지입니다. </li></ol>

<ol>
	<li>Webb, S. E., Miller, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signalling+during+embryonic+development.">Calcium signalling during embryonic development.</a> Nat Rev Mol Cell Biol. 4, 539-551 (2003).</li><li>Webb, S. E., Miller, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++signalling+and+early+embryonic+patterning+during+zebrafish+development.">Ca2+ signalling and early embryonic patterning during zebrafish development.</a> Clin Exp Pharmacol Physiol. 34, 897-904 (2007).</li><li>Whitaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+at+fertilization+and+in+early+development.">Calcium at fertilization and in early development.</a> Physiol. Rev. 86, 25-88 (2006).</li><li>Yuen, M. Y., 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=Characterization+of+Ca(2+)+signaling+in+the+external+yolk+syncytial+layer+during+the+late+blastula+and+early+gastrula+periods+of+zebrafish+development.">Characterization of Ca(2+) signaling in the external yolk syncytial layer during the late blastula and early gastrula periods of zebrafish development.</a> Biochim Biophys Acta. 1833, 1641-1656 (2013).</li><li>Tombes, R. M., Borisy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+free+calcium+and+mitosis+in+mammalian+cells:+anaphase+onset+is+calcium+modulated,+but+is+not+triggered+by+a+brief+transient.">Intracellular free calcium and mitosis in mammalian cells: anaphase onset is calcium modulated, but is not triggered by a brief transient.</a> J. Cell Biol. 109, 627-636 (1989).</li><li>Hudmon, A., Schulman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+Ca2+/Calmodulin-Dependent+Protein+Kinase+II:+The+Role+of+Structure+and+Autoregulation+in+Cellular+Function.">Neuronal Ca2+/Calmodulin-Dependent Protein Kinase II: The Role of Structure and Autoregulation in Cellular Function.</a> Annu. Rev. Biochem. 71, 473-510 (2002).</li><li>Tombes, R. M., Faison, M. O., Turbeville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+Evolution+of+Multifunctional+Ca2+/CaM-dependent+Protein+Kinase+(CaMK-II).">Organization and Evolution of Multifunctional Ca2+/CaM-dependent Protein Kinase (CaMK-II).</a> Gene. 322, 17-31 (2003).</li><li>Braun, A. P., Schulman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Multifunctional+Calcium/Calmodulin-Dependent+Protein+Kinase:+From+Form+to+Function.">The Multifunctional Calcium/Calmodulin-Dependent Protein Kinase: From Form to Function.</a> Annu. Rev. Physiol. 57, 417-445 (1995).</li><li>Rich, R. C., Schulman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate-directed+function+of+calmodulin+in+autophosphorylation+of+Ca2+/calmodulin-dependent+protein+kinase+II.">Substrate-directed function of calmodulin in autophosphorylation of Ca2+/calmodulin-dependent protein kinase II.</a> J Biol Chem. 273, 28424-28429 (1998).</li><li>Rothschild, S. C., 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=Tbx5-mediated+expression+of+Ca2+/calmodulin-dependent+protein+kinase+II+is+necessary+for+zebrafish+cardiac+and+pectoral+fin+morphogenesis.">Tbx5-mediated expression of Ca2+/calmodulin-dependent protein kinase II is necessary for zebrafish cardiac and pectoral fin morphogenesis.</a> Dev Biol. 330, 175-184 (2009).</li><li>Rothschild, S. C., Lister, J. A., Tombes, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+CaMK-II+genes+during+early+zebrafish+embryogenesis.">Differential expression of CaMK-II genes during early zebrafish embryogenesis.</a> Dev Dyn. 236, 295-305 (2007).</li><li>Francescatto, L., Rothschild, S. C., Myers, A. L., Tombes, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+activation+of+membrane+targeted+CaMK-II+in+the+zebrafish+Kupffer's+vesicle+is+required+for+left-right+asymmetry.">The activation of membrane targeted CaMK-II in the zebrafish Kupffer's vesicle is required for left-right asymmetry.</a> Development. 137, 2753-2762 (2010).</li><li>Rothschild, S. C., Francescatto, L., Drummond, I. A., Tombes, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMK-II+is+a+PKD2+target+that+promotes+pronephric+kidney+development+and+stabilizes+cilia.">CaMK-II is a PKD2 target that promotes pronephric kidney development and stabilizes cilia.</a> Development. 138, 3387-3397 (2011).</li><li>Rothschild, S. C., 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=CaMK-II+activation+is+essential+for+zebrafish+inner+ear+development+and+acts+through+Delta-Notch+signaling.">CaMK-II activation is essential for zebrafish inner ear development and acts through Delta-Notch signaling.</a> Dev Biol. 381, 179-188 (2013).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos.">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. , e1113 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. , e1115 (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book: A guide for the laboratory use of zebrafish (Brachydanio rerio). , (1993).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li><li>Obara, T., 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=Polycystin-2+immunolocalization+and+function+in+zebrafish.">Polycystin-2 immunolocalization and function in zebrafish.</a> J Am Soc Nephrol. 17, 2706-2718 (2006).</li><li>Chitramuthu, B. P., Bennett, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+whole+mount+in+situ+hybridization+within+zebrafish+embryos+to+study+gene+expression+and+function.">High resolution whole mount in situ hybridization within zebrafish embryos to study gene expression and function.</a> J Vis Exp. , e50644 (2013).</li><li>Thisse, C., Thisse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+in+situ+hybridization+to+whole-mount+zebrafish+embryos.">High-resolution in situ hybridization to whole-mount zebrafish embryos.</a> Nature. 3, 59-69 (2008).</li><li>Harris, P., Osborn, M., Weber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+tubulin-containing+structures+in+the+egg+of+the+sea+urchin+Strongylocentrotus+purpuratus+from+fertilization+through+first+cleavage.">Distribution of tubulin-containing structures in the egg of the sea urchin Strongylocentrotus purpuratus from fertilization through first cleavage.</a> J Cell Biol. 84, 668-679 (1980).</li></ol>

저자는 더 경쟁 재정적 이해 관계가 없음을 선언합니다.

PFA / 메탄올 방법은 제브라 피쉬 개발하는 동안 포스-T 287 -CaMK-II 에피토프의의 면역을 최적화의 주요 목적이 실험실에서 개발되었다. 이 방법은 성공적으로 제브라 피쉬의 KV, 14, 특히 KV 단계에서 신장. (13),이 기술이 필요했다 (12) 내이 등 여러 기관 섬모의 형성 동안 P-CaMK-II를 현지화. 이러한 방법의 성공으로 인해자가 형광의) 최소화, 포스 CaMK-II 에피토프 b) 보존...

The techniques described here are the outcome of studies that have sought to define downstream targets of Ca2+ signals during events that occur during early development, including fertilization, gastrulation, somitogenesis and trunk, eye, brain and organ formation.1-3 The original discoveries of embryonic Ca2+ signaling were dependent on the use of natural and engineered Ca2+ indicators, such as aequorin4 and fura-2.5 Even with current technology, the detection of transient elevations of Ca2+ requires cumbersome analytical tools and does not reveal the targets of such Ca2+ signals. 
This laboratory investigates Ca2+ signals that act through the Ca2+/calmodulin-dependent (multifunctional) protein kinase known as CaMK-II, an enzyme that is enriched in the central nervous system and originally identified as a regulator of long-term potentiation.6 CaMK-II is not brain-specific, is widely expressed and highly conserved throughout the entire lifespan and bodies of species throughout the animal kingdom, including invertebrates.7,8 CaMK-II has the unique capability of sustaining its own activity even after Ca2+ levels have diminished due to its ability to autophosphorylate at Thr287. In this autophosphorylated state, CaMK-II remains active in a Ca2+/CaM-independent manner, until dephosphorylated.6 Thus, the localization of phosphorylated CaMK-II (Thr287) can identify cells in which natural, relevant Ca2+ elevations have occurred. 
An antibody against autophosphorylated (P-Thr287) mammalian CaMK-II has been well-characterized and was initially used to localize activated CaMK-II in brain tissue.9 Zebrafish (Danio rerio) have seven CaMK-II genes10,11 whose protein products contain a sequence of MHRQE[pT287]VECLK in this region.10,11 This sequence is very similar to the phosphopeptide antigen used to create this rabbit polyclonal antibody (MHRQE[pT]VDCLK; Upstate/Millipore) and therefore it was not a complete surprise that this antibody cross-reacted with zebrafish CaMK-II. This laboratory showed that this antibody reacts with zebrafish CaMK-II in proportion to autophosphorylation and Ca2+/CaM-independent activity.12 Additional pan-specific CaMK-II antibodies have also been shown to cross-react with zebrafish CaMK-II.13
This antibody has been used to demonstrate that zebrafish CaMK-II is preferentially activated in cells on one side of the zebrafish Kupffer's Vesicle (KV), the ciliated organ necessary for establishment of left/right asymmetry.12 This antibody was used to demonstrate that CaMK-II is transiently activated in four adjacent cells on the left side of the KV during the exact same developmental phase that organ positioning is determined.12 In addition to the Kupffer's Vesicle (KV), autophosphorylated (P-T287) was also located in specific intracellular sites in other ciliated tissues including the kidney, neuromasts, and inner ear.12,13 In the zebrafish kidney, P-T287-CaMK-II is enriched along the apical border of ciliated ductal cells and within cloacal cilia where it influences their assembly.13 Finally, in the developing inner ear, P-T287-CaMK-II is intensely concentrated at the base of cilia and influences cell differentiation through the Delta-Notch signal pathway.14 In summary, the detection of activated CaMK-II has pinpointed sites of intracellular Ca2+ release and illuminated potential new signaling pathways.
These discoveries were completely dependent on developing a sensitive and accurate method to localize activated (P-T287-autophosphorylated) CaMK-II. The methods to fix and immunostain the zebrafish KV, kidney and inner ear are described. The limitations of this technique are also described. These techniques should be useful to any investigator who seeks to obtain high-resolution images in two fluorescent channels of not just phospho-epitopes, but any epitope, during early vertebrate development.

<table border="0" cellpadding="0" cellspacing="0" width="886">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">1-phenyl-2-thiourea (PTU)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">P-7629</td>
			<td style="width:347px;">0.12% Stock solution. Dilute 1:40 in system water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa488 anti-mouse IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11001</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa488 anti-rabbit IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11008</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa488 phalloidin</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A12379</td>
			<td>Preferentially binds to F-actin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa568 anti-mouse IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11004</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa568 anti-rabbit IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11011</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-acetylated a-tubulin</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">T7451</td>
			<td style="width:347px;">Mouse monoclonal, use at 1:500</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:247px;">anti-phospho-T287 CaMK-II</td>
			<td style="width:175px;">EMD Millipore</td>
			<td style="width:119px;">06-881</td>
			<td style="width:347px;">Rabbit polyclonal, use at 1:20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-total CaMK-II</td>
			<td style="width:175px;">BD Biosciences</td>
			<td style="width:119px;">611292</td>
			<td style="width:347px;">Mouse monoclonal, use at 1:20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ethanol</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">S96857</td>
			<td style="width:347px;">Lab grade, 95% denatured</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Forceps</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td>11252-20</td>
			<td>Dumont #5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glass coverslips</td>
			<td style="width:175px;">VWR</td>
			<td style="width:119px;">16004-330</td>
			<td>#1&#160; thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glass microscope slides</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">12-550-15</td>
			<td>Standard glass slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Methanol</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">A411</td>
			<td>Store in freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Microcentrifuge tubes</td>
			<td style="width:175px;">VWR</td>
			<td style="width:119px;">20170-038</td>
			<td>capped tubes, not sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Normal goat serum</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">16210-064</td>
			<td>Aliquot 1ml tubes, store in freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Paraformaldehyde</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">P-6148</td>
			<td>Reagent grade, crystalline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Phosphate buffered saline (PBS)</td>
			<td style="width:175px;">Quality Biological</td>
			<td style="width:119px;">119-069-131</td>
			<td>10X stock solution or made in lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Triton X-100</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">BP-151</td>
			<td>10% solution in water, store at room temp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tween-20</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">85113</td>
			<td>10% solution in water, store at room temp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Compound microscope</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:119px;">E-600</td>
			<td>Mount on vibration-free table</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">C1 Plus two-laser scanning confocal</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:119px;">C1 Plus</td>
			<td>Run by EZ-C1 program, but upgrades use &#34;Elements&#34;</td>
		</tr>
	</tbody>
</table>

immunostaining phospho-epitopes in ciliated organs of whole mount zebrafish embryos

세포의 급속한 확산, 유전자의 조직 - 특이 적 발현 및 시그널링 네트워크의 출현은 모든 척추 동물의 초기 배아 발달의 특성. 심지어 하나의 셀 내에서 - - 반응 속도 및 신호의 위치 배아의 중요한 발달 유전자의 식별을 보완합니다. 면역 염색 기술이 차 섬모 작게 구조의 세포 내 및 전체 동물 신호의 동역학을 정의하기 위해 도시되었다는 것을 설명한다. 레이저 스캐닝 공 초점 복합 현미경을 사용하여 정착, 영상 및 이미지 처리 기술은 적은 36 시간 내에 완료 될 수있다. 제브라 피쉬 (다니오 레 리오)는 저렴하고 인간의 질병에 관련된 척추 동물 종에서 연구를 수행하고자 연구자를위한 바람직한 유기체이다. 유전자 녹아웃 또는 knockdowns는 실제 단백질 제품의 손실에 의해 확인되어야한다. 단백질 손실 이러한 확인여기에 설명 된 기술을 사용하여 달성 될 수있다. 신호 전달 경로에 대한 단서는 번역 후 인산화에 의해 변형 된 단백질과 반응하는 항체를 사용하여 해독 할 수 있습니다. 보존 및 에피토프의 인산화 상태를 최적화하는 것은이 결정하는 것이 중요하며,이 프로토콜에 의해 달성된다. 이 연구는 개발의 첫 72 시간 동안 배아를 해결하기 위해 기술을 설명 쿠퍼의 소포 (KV)의 섬모와 관련 항원의 다양한 신장과 내이 공동 현지화. 이러한 기술은 절개하지 않아도, 간단하고 비교적 짧은 시간에 완료 될 수있다. 하나의 이미지로 공 초점 이미지 스택을 투사하는 것은 이러한 데이터를 제시하는 유용한 수단이다.

포스 에피토프를 시각화하기위한 최적의 조건 제브라 피쉬 배아에서 단백질 항원 결정기의의 면역을 설명하는 방법은 현장 하이브리드의 mRNA를 통해 현지화에 대한에 비해 상대적으로 드문 드문 있었다. 제브라 피쉬 배아의 섬모 세포에서 단백질 에피토프를 로컬 라이즈에 사용되는 고정 제 4 % PFA / PBS 및...

Watch this Scientific Journal Video about Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos at JoVE.com

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

기술은 전체 제브라 피쉬 배아에서 포스 항원을 발현 사이 다음 주 섬모와 같은 작은 세포 구조에서 두 색 형광 공 초점 현지화를 수행 할 수 있도록 서술되어있다. 고정 및 이미징 기술은 위치 특정 단백질의 모양이나 활성화 동력학을 정의 할 수있다.

전체 마운트 Zebrafish의 태아의 섬모 기관에서 면역 염색 포스 - 항원 결정기

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

immunostaining-phospho-epitopes-ciliated-organs-whole-mount-zebrafish

Research

JoVE Journal

Chemistry

7.9K 조회수.  Virginia Commonwealth University.  기술은 전체 제브라 피쉬 배아에서 포스 항원을 발현 사이 다음 주 섬모와 같은 작은 세포 구조에서 두 색 형광 공 초점 현지화를 수행 할 수 있도록 서술되어있다. 고정 및 이미징 기술은 위치 특정 단백질의 모양이나 활성화 동력학을 정의 할 수있다. 

비디오: Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn and 70Zn) which are naturally fractionated between mouse organs. We first show how to dissolve the different organs in order to free the Zn atoms; this step is realized by a mixture of HNO3 and H2O2. We then purify the zinc atoms from all the other elements, in particular from isobaric interferences (e.g., Ni), by anion-exchange chromatography in a dilute HBr/HNO3 medium. These first two steps are performed in a clean laboratory using high purity chemicals. Finally, the isotope ratios are measured by using a multi-collector inductively-coupled-plasma mass-spectrometer, in low resolution. The samples are injected using a spray chamber and the isotopic fractionation induced by the mass-spectrometer is corrected by comparing the ratio of the samples to the ratio of a standard (standard bracketing technique).&#160;This full typical&#160;procedure produces an isotope ratio with a 50 ppm (2 s.d.) reproducibility.

We present the technique to measure with high precision zinc isotope ratios in mouse organs.

마우스 기관에 적용 높은 정밀도 아연 동위 원소 측정

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn ...

연구

화학

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

고성능 액체 크로마토 그래피 결합 흐름 섬광에 의한 방사성 표지 및 Phosphoinositides의 세포 수준의 정량화

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Accessing Valuable Ligand Supports for Transition Metals: A Modified, Intermediate Scale Preparation of 1,2,3,4,5-Pentamethylcyclopentadiene

A reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented, based on modifications of existing protocols that derive from initial 2-bromo-2-butene lithiation followed by acid mediated dienol cyclization. The revised synthesis and purification of the ligand avoids the use of mechanical stirring while still permitting access to significant quantities (39 g) of Cp*H in good yield (58%). The procedure offers other additional benefits, including a more controlled quench of excess lithium during the production of the intermediate heptadienols and a simplified isolation of Cp*H of sufficient purity for metallation with transition metals. The ligand was subsequently used to synthesize [Cp*MCl2]2 complexes of both iridium and ruthenium to demonstrate the utility of the Cp*H prepared and purified by our method. The procedure outlined herein affords substantial quantities of a ubiquitous ancillary ligand support used in organometallic chemistry while minimizing the need for specialized laboratory equipment, thus providing a simpler and more accessible entry point into the chemistry of 1,2,3,4,5-pentamethylcyclopentadiene.

Reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented. The revised protocol for the synthesis and purification of the ligand minimizes the need for specialized laboratory equipment while simplifying reaction workups and product purification. Use of Cp*H in the synthesis of [Cp*MCl2]2 complexes (M = Ru, Ir) is also described.

가치있는 리간드에 액세스하면 전이 금속에 대한 지원 : 1,2,3,4,5-Pentamethylcyclopentadiene의 수정, 중간 규모 준비

A reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented, based on modifications of existing protocols that ...

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

단일 셀 대사체학 라이브 개구리 (Xenopus laevis) 배아에 대 한 현미경 모 세관 전기 이동 법 질량 분석

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

F− 의 310 Nm 분리에 의해 설명 하는 음이온의 광전자 이미징

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

Bio-inspired Polydopamine Surface Modification of Nanodiamonds and Its Reduction of Silver Nanoparticles

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a simple protocol for the multifunctional surface modification of NDs by using mussel-inspired polydopamine (PDA) coating. In addition, the functional layer of PDA on NDs could serve as a reducing agent to synthesize and stabilize metal nanoparticles. Dopamine (DA) can self-polymerize and spontaneously form PDA layers on ND surfaces if the NDs and dopamine are simply mixed together. The thickness of a PDA layer is controlled by varying the concentration of DA. A typical result shows that a thickness of ~5 to ~15 nm of the PDA layer can be reached by adding 50 to 100 &#181;g/mL of DA to 100 nm ND suspensions. Furthermore, the PDA-NDs are used as a substrate to reduce metal ions, such as Ag[(NH3)2]+, to silver nanoparticles (AgNPs). The sizes of the AgNPs rely on the initial concentrations of Ag[(NH3)2]+. Along with an increase in the concentration of Ag[(NH3)2]+, the number of NPs increases, as well as the diameters of the NPs. In summary, this study not only presents a facile method for modifying the surfaces of NDs with PDA, but also demonstrates the enhanced functionality of NDs by anchoring various species of interest (such as AgNPs) for advanced applications.

A facile protocol is presented to functionalize the surfaces of nanodiamonds with polydopamine.

Nanodiamonds 및은 나노 입자의 그것의 감소의 바이오 영감 Polydopamine 표면 수정

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

합성 및 Bioconjugation의 Thiol 반응 시 약 사이트에 선택적으로의 창조에 대 한 수정 Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

화합물 억제, IC50 값, 관절 활성 및 뉴클레오시드 리보하이드라제의 전세포 활성을 결정하기 위한 NMR 기반 활성 반응

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...