우리는 도움이 덧글에 대한 A. 이럴 감사하고 원고를 읽을 수 있습니다. 이 작품은 셀 단위로 생물 의학 연구위원회 (MRC) 기금에 의해 그리고 인간 프론티어 과학 프로그램 그랜트 (RGP0048/2009-C)에 의해 지원되었다.

1. 효소 반응 - 1 일 (오후에 1 시간) <ol><li> 첫 번째 단계는 IP 6 K1이나 VIP1이 pyrophosphorylated isoforms에 IP 6으로 변환하는 효소 반응을 독립 10-20를 준비하는 것입니다. </li><li> 우리는 E.에서 정화 그의 - IP 6 K1과 GST - Vip1 효소를 사용하여 프로토콜에 따라 대장균 이전 17,18 설명했다. </li><li> 1X 반응 완충액 (30 MM의 Hepes 산도 6.8, 50 MM NaCl, 6 MM MgSO 4, 1 ㎜ DTT), 6 당연 PhosphoCreatine (PCR), 25 U / ML CreatinePhosphoKinase (CPK), 5 MM ATP (MG가 포함된 50 μL 반응 준비 소금), 0.3 MM IP6, 0.05-0.1 μg 그의 - IP 6 K1이나 GST - Vip1. MiliQ ddH 2 O.과 볼륨을 조정 </li><li> 간단히 37 반응과 부화 ° C 하루 회전을 스핀. </li></ol> 2. Polyacrylamide 젤 주조 및 로딩 - 2 일 (오후 4 시간) <ol><li> polyacrylamide 젤은 24cm 길이 18cm 폭 유리 접시와 1.5 mm 폭 스페이서를 사용하여 준비가되어 있습니다. 보통 16 차선 또는 예비 하나의 레인 빗이 사용됩니다. </li><li> 35.5 % (W / V) 아크릴 아미드 : 비스 - 아크릴 아미드 19시 1분, 1X 트리스 / Borate / EDTA (에틸렌 다이아 민 테트라 초산) (TBE), 0.05 % (W / V) 암모늄 persulfate (APS는 다음을 포함하는 혼합 (50 ML / 겔) 준비 ), 0.05 % (W / V)가 Temed. , 사전 c​​asted 유리 플레이트 사이의 믹스를 부어 빗를 넣고 RT에서 30~60분을위한 중합 보자. </li><li> 젤 polymerized되면​​ 추위 방기구를 전송하고 200-300 볼트에서 30-60 분 동안 1X TBE에 미리 실행합니다. </li><li> 각 반응 OrangeG 염료의 1X (10 MM 트리스 - HCL pH7.0, 1 MM EDTA (에틸렌 다이아 민 테트라 초산), 30 % 글리세롤, 0.1 % OrangeG) 추가합니다. 표준 컨트롤로 로드할 수 IP 6 2 nmol을 포함하는 샘플을 준비합니다. </li><li> 주사기 및 침전물을 제거하는 21G 바늘을 사용하여 버퍼를 실행하는 각 잘 철저히 세척 후 젤을로드합니다. 측면 우물에 로딩하지 마십시오. </li><li> OrangeG 염료 밴드가 젤 하단에서 마지막 10cm 이내가 될 때까지, 450-550 볼트 (7 mAmp / 겔)에서 하룻밤 젤을 실행합니다. </li></ol> 3. IP 7 절연 - 3 일 (4 시간)과 일 4 (6-7시간의 SpeedVac 건조 과정) <ol><li> 겔 기기를 분해 조심스럽게 다른 하나 젤 떠나는 한 유리 플레이트를 제거합니다. 그림 1에 표시된 IP 6 표준 하나의 샘플 레인이있는 바닥에 그냥 OrangeG 염료 대역 이상에서 젤의 일부를 잘라. </li><li> 컷 몇 분 동안 Toluidine 블루 (0.1 % (W / V) toluidine 파란색, 20 % (W / V) 메탄올, 2 % (W / V) 글리세롤)로 젤의 부분 (1-3 분) 또는 스테인 이노시톨 나트륨 밴드가 나타날 때까지. 이전에 건조에서 흠없는 젤을 방지하기 위해 젤 위에 다시 제거 유리 접시 놔. </li></ol> 그것은 IP 6 표준보다 약간 느리게 동작하기 때문에 IP 7 밴드가 표시됩니다. IP 6보다 빠르게 실행 ATP는 또한 (그림 1) 표시되어야합니다. 몇 분 동안 데 - 얼룩 솔루션 (20 % (W / V) 메탄올)에 젤의 스테인드 부분을 전송, Toluidine 블루의 초과 멀리 세척하고 흠없는 젤로 젤을 조정. 높은 pyrophosphorylated 이노시톨 isoforms (IP 8 IP 9)의 시각화가 필요한 경우, 상온에서 20 분 Toluidine 블루 얼룩 솔루션 젤 얼룩. 그 후, 약 15 분 드 얼룩 솔루션 Toluidine의 블루 멀리 씻으십시오. <ol start="3"><li> 면도날은 참조로 스테인드 젤 (그림 1)로 결정 IP 7 마이 그 레이션하는 위치를 사용하여 젤의 흠없는 부분에 IP 7 밴드를 잘라 함께. </li><li> 15 ML 튜브의 겔에서 절단했던 IP 7 밴드 넣고 MilliQ ddH 2 O. 10 ML을 추가 실온에서 10 분 순환에 튜브를 넣어. TBE와 미세한 입자의 아크릴 아미드 초과 제거하는 액체를 폐기하십시오. </li><li> 그 후, 두 탈수 - 수화 사이클을 수행합니다. IP에 7을 포함하는 젤로 튜브의 50 % (W / V) 메탄올 5 ML을 추가하고 2 시간 동안 실온에서 회전. MilliQ ddH 2 O 5 ML를 포함하는 새로운 15 ML 튜브로 겔 슬라이스를 전송하고 2 시간 동안 실온에서 회전. 튜브에서 메탄올과 MilliQ H 2 O을 삭제하지 마십시오. 이전에 사용한 15 ML 튜브에 다시 전송 polyacrylamide 젤로 한 번 더 탈수 - 수화주기를 반복합니다. 세척 중 하나는 밤을 통해 수행할 수 있습니다. </li><li> eluted IP 7을 집중, 60 온수 SpeedVac ° C.를 사용하여 (5 ML MilliQddH 2 O 5 ML 50 % (W / V) 메탄올) 함께 10 ML을 건조 </li><li> 샘플이 거의 건조되면 a1.5 ML의 원심 분리기 튜브 및 5000 rpm으로 2 분 동안 회전하는 나머지 액체 (300-600 μl)를 전송합니다. </li><li> 새로운 1.5 ML의 원심 관에 뜨는 및 전송 수집, 휴가이 아크릴 아미드 입자를 포함할 수 있으므로 하단 20-30 μL. </li><li> 필요한 경우하면 unheated Speed​​Vac를 사용하여 건조 과정을 계속합니다. 샘플 100-300 μL의 볼륨에 도달하면 샘플이 완전히 건조하는 경우 IP 7 복구가 급격하게 감소, 따라서 건조 과정을 종료할 수 있습니다. </li></ol> 4. IP 7 농도 및 순도의 결정. <ol><li> 마찬가지로 제 2.2-2.5로, 페이지 젤에서 실행 회수 IP 7 샘플 2-5 μL를 사용하십시오. 농도 표준 및 폴리 - P 마커 4 nmol으로 IP 6 여러 dilutions (예 : 0.5, 1, 2, 4 nmol)을로드합니다. 젤을 실행 후, 섹션 3.2 (그림 2A)에 설명된 절차에 따라 얼룩 및 Toluidine 블루 솔루션 드 얼룩 전체 젤로 이노시톨 나트륨의 isoforms을 시각화. </li><li> Toluidine의 얼룩 후, 농도는 그림의 2B에 나타난 젤 검사 및 IP 6 IP 7 일 사이 강도의 차이를 비교, 같은 이미지 - J와 같은 이미징 소프트웨어를 사용하여 결정하실 수 있습니다. </li></ol> 5. 대표 결과 : IP 6 K1과 VIP1 효소를 사용하여 IP 7 IP 6 예비 효소 변환은 쉽게 페이지 분석 (그림 1)를 사용하여 해결될 수 있습니다. 그들은 이노시톨 고리에 존재하는 인산염 그룹의 수에 따라 느리게 실행 이후 Toluidine 블루 겔 얼룩과 함께 크기 조절과 같은 IP 6 로딩은 pyrophosphorylated derivates의 확인하실 수 있습니다. 위에서 설명한 절차는 IP 7 쉽게 정화 수 있습니다. 페이지에서 정화 이노시톨 나트륨의 분석은 우리의 IP 7 (그림 2A)의 순결을 공개했다. 흥미롭게도, VIP1의 IP 7 제품의 1/3PP-IP 5 이성질체는 IP 6 K1에 의해 생성되는 IP 7 5PP - IP 5 이성질체보다 약간 느린 마이 그 레이션합니다. 정화 IP 7 (그림 2B)의 농도 쉽게 부량을 허용 IP 6 기준 중 하나를 사용하십시오. 자세한 실험 IP 7을 사용하기 전에, 그 생물 학적 활동은 평가 수 (그림 3). 5PP - IP 5 VIP1과 IP 7 인산 가수 분해 효소 DDP1 (diphosphoinositol의 polyphosphate의 phosphohydrolase)와 incubated입니다. 정기적으로, 정화 IP 7 DDP1 (그림 3)에 의해 VIP1하여 IP 8과 IP 6으로 변환됩니다. <img alt="그림 1" src="/files/ftp_upload/3027/3027fig1.jpg" /> 그림 1 :. IP 7 밴드의 페이지와 고립의 Toluidine 얼룩은 표준 IP (6)이있는 젤의 부분은 절단 및 Toluidine 블루 솔루션을 사용하여 얼룩되었습니다. 세 밴드가 (위에서 아래로) IP 7, IP 6 ATP를 나타냅니다. 젤의 스테인드 부분은 다음 젤의 나머지에 부합했다. 이것은 다음 (점선 상자) 잘라 정화 수있는 IP의 7을 포함하는 젤 부분의 현지화 수 있습니다. <img alt="그림 2" src="/files/ftp_upload/3027/3027fig2.jpg" /> 그림 2 : IP 6 K1과 VIP1 반응 제품의 페이지 분석. A) Toluidine 블루 염색법에 의해 IP 6 분석 (4, 2, 1, 0.5 nmol)는 IP 6 K1 (5PP - IP 5) 및 ​​VIP1 (1/3PP-IP 모두에서 정화 IP 7 농도를 결정하기 위해 사용되었습니다 5) 반응. B) 스캐터 플롯 분석 정화 IP 7의 농도를 결정합니다. 농도는 IP 6 미리 정해진 금액에 비해 imageJ 소프트웨어를 사용하여 계산 밴드 강도,에 따라 결정됩니다. X - 축은 농도를 나타내고 y 축은 nmol 표현 농도를 나타냅니다. <img alt="그림 3" src="/files/ftp_upload/3027/3027fig3.jpg" /> 그림 3 :. IP 7 생물 학적 활동의 분석 정화 IP 7의 품질을 확인하려면 우리는 DDP1 VIP1 또는 IP 7 인산 가수 분해 효소와 5PP - IP 5 (IP 6 K1 생성된 IP 7) incubated 다음 페이지에 대한 반응을 해결. DAPI와 Toluidine 얼룩이 VIP1의 IP 8 예상 생산과 DDP1하여 IP 6 IP 7의 변환을 공개했다.

<ol>
	<li>Bennett, M., Onnebo, S. M., Azevedo, C., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates:+metabolism+and+signaling.">Inositol pyrophosphates: metabolism and signaling.</a> Cell Mol Life Sci. 63, 552-564 (2006).</li><li>Burton, A., Hu, X., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+inositol+pyrophosphates+signalling+molecules?.">Are inositol pyrophosphates signalling molecules?.</a> J Cell Physiol. 220, 8-15 (2009).</li><li>Barker, C. J., Illies, C., Gaboardi, G. C., Berggren, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates:+structure,+enzymology+and+function.">Inositol pyrophosphates: structure, enzymology and function.</a> Cell Mol Life Sci. 66, 3851-3871 (2009).</li><li>Shears, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diphosphoinositol+polyphosphates:+metabolic+messengers.">Diphosphoinositol polyphosphates: metabolic messengers.</a> Mol Pharmacol. 76, 236-252 (2009).</li><li>Saiardi, A., Erdjument-Bromage, H., Snowman, A. M., Tempst, P., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+diphosphoinositol+pentakisphosphate+by+a+newly+identified+family+of+higher+inositol+polyphosphate+kinases.">Synthesis of diphosphoinositol pentakisphosphate by a newly identified family of higher inositol polyphosphate kinases.</a> Curr Biol. 9, 1323-1326 (1999).</li><li>Draskovic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+hexakisphosphate+kinase+products+contain+diphosphate+and+triphosphate+groups.">Inositol hexakisphosphate kinase products contain diphosphate and triphosphate groups.</a> Chem Biol. 15, 274-286 (2008).</li><li>Mulugu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+family+of+enzymes+that+phosphorylate+inositol+hexakisphosphate.">A conserved family of enzymes that phosphorylate inositol hexakisphosphate.</a> Science. 316, 106-109 (2007).</li><li>Fridy, P. C., Otto, J. C., Dollins, D. E., York, 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=Cloning+and+characterization+of+two+human+VIP1-like+inositol+hexakisphosphate+and+diphosphoinositol+pentakisphosphate+kinases.">Cloning and characterization of two human VIP1-like inositol hexakisphosphate and diphosphoinositol pentakisphosphate kinases.</a> J Biol Chem. 282, 30754-30762 (2007).</li><li>Illies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+inositol+pyrophosphates+for+full+exocytotic+capacity+in+pancreatic+beta+cells.">Requirement of inositol pyrophosphates for full exocytotic capacity in pancreatic beta cells.</a> Science. 318, 1299-1302 (2007).</li><li>Saiardi, A., Resnick, A. C., Snowman, A. M., Wendland, B., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+regulate+cell+death+and+telomere+length+through+phosphoinositide+3-kinase-related+protein+kinases.">Inositol pyrophosphates regulate cell death and telomere length through phosphoinositide 3-kinase-related protein kinases.</a> Proc Natl Acad Sci U S A. 102, 1911-1914 (2005).</li><li>York, S. J., Armbruster, B. N., Greenwell, P., Petes, T. D., York, 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=Inositol+diphosphate+signaling+regulates+telomere+length.">Inositol diphosphate signaling regulates telomere length.</a> J Biol Chem. 280, 4264-4269 (2005).</li><li>Luo, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+mediate+chemotaxis+in+Dictyostelium+via+pleckstrin+homology+domain-PtdIns(3,4,5)P3+interactions.">Inositol pyrophosphates mediate chemotaxis in Dictyostelium via pleckstrin homology domain-PtdIns(3,4,5)P3 interactions.</a> Cell. 114, 559-572 (2003).</li><li>Saiardi, A., Sciambi, C., McCaffery, J. M., Wendland, B., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+regulate+endocytic+trafficking.">Inositol pyrophosphates regulate endocytic trafficking.</a> Proc Natl Acad Sci U S A. 99, 14206-14211 (2002).</li><li>Auesukaree, C., Tochio, H., Shirakawa, M., Kaneko, Y., Harashima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plc1p,+Arg82p,+and+Kcs1p,+enzymes+involved+in+inositol+pyrophosphate+synthesis,+are+essential+for+phosphate+regulation+and+polyphosphate+accumulation+in+Saccharomyces+cerevisiae.">Plc1p, Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae.</a> J Biol Chem. 280, 25127-25133 (2005).</li><li>Azevedo, C., Burton, A., Ruiz-Mateos, E., Marsh, M., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphate+mediated+pyrophosphorylation+of+AP3B1+regulates+HIV-1+Gag+release.">Inositol pyrophosphate mediated pyrophosphorylation of AP3B1 regulates HIV-1 Gag release.</a> Proc Natl Acad Sci U S A. 106, 21161-21166 (2009).</li><li>Bhandari, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+pyrophosphorylation+by+inositol+pyrophosphates+is+a+posttranslational+event.">Protein pyrophosphorylation by inositol pyrophosphates is a posttranslational event.</a> Proc Natl Acad Sci U S A. 104, 15305-15310 (2007).</li><li>Azevedo, C., Burton, A., Bennett, M., Onnebo, S. M., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+InsP7+by+the+Inositol+Hexakisphosphate+Kinase+1+(IP6K1).">Synthesis of InsP7 by the Inositol Hexakisphosphate Kinase 1 (IP6K1).</a> Methods Mol Biol. 645, 73-85 (2010).</li><li>Otto, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+analysis+of+inositol+phosphate+kinases.">Biochemical analysis of inositol phosphate kinases.</a> Methods Enzymol. 434, 171-185 (2007).</li><li>Losito, O., Szijgyarto, Z., Resnick, A. C., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+and+their+unique+metabolic+complexity:+analysis+by+gel+electrophoresis.">Inositol pyrophosphates and their unique metabolic complexity: analysis by gel electrophoresis.</a> PLoS One. 4, e5580-e5580 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

생화학에서 이노시톨 나트륨의 사용은 심각하게 이러한 화합물의 상업 unavailability와 기존의 검색 방법의 가난한 감도에 의해 제한됩니다. 다른 인산염 그룹의 번호, Toluidine 블루 (그림 1), 인산염 그룹에 바인딩 metachromatic 염료를 가진 분자의 분리를 가능하게 페이지의 결합 연구 20 새로운 길을 여는 이노시톨 pyrophoshate의 isoforms의 쉬운 검색이 가능합니다. 효소 반응의 이노시톨 나트륨 제품을 정화 페이지 기술의 설명 사용 IP 6 K1이나 VIP1 하나 고품질의 IP 7 다량의 생산을 허용 간단, 경제적 안정 방법입니다 outby 실시. 위에서 설명한 방법은 7 IP의 간단한 정화에 국한되지 않습니다하지만 설명 프로토콜의 사소한 수정 이노시톨 pyrophosphates의 다른 범위의 정화를 허용할 수 있습니다. 이상의 여덟 인산염 그룹을 포함하는 높은 phosphorylated 이노시톨 나트륨의 isoforms은 기판 20,6로 IP 7 IP 6 다른 양의를 사용하여 검색하실 수 있습니다. 이러한 이노시톨 pyrophosphates은 얼룩 절차의 길이를 증가하고 이후 (3.2) 정화에 의해 감지하실 수 있습니다. 또한, 효소 반응을위한 기판으로 IP 5 사용 PP - IP 5 정화 및 이노시톨 고리에 수산기 그룹을 포함하는 다른 이노시톨 pyrophosphates을 허용합니다. 결론적으로,이 undemanding 방법은 이처럼 흥미로운 연구 분야에 대한 새로운 길을 열어, 널리 사용 악기 이노시톨 pyrophosphates의 밀리그램 수량의 안정적인 정화를 위해 수 있습니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> Phytic의 산성 (IP 6) </td><td> 시그마 - 알드리치 </td><td> P8810 </td></tr><tr><td> 폴리 - P (나트륨 hexametaphosphate) </td><td> 시그마 - 알드리치 </td><td> P8510 </td></tr><tr><td> ATP - MG 2 + 소금 </td><td> 시그마 - 알드리치 </td><td> A9187 </td></tr><tr><td> OrangeG </td><td> 시그마 - 알드리치 </td><td> O3756 </td></tr><tr><td> PhosphoCreatine (PCR) </td><td> 시그마 - 알드리치 </td><td> P7936 </td></tr><tr><td> CreatinePhospho의 키나제 (CPK) </td><td> 시그마 - 알드리치 </td><td> C3755 </td></tr><tr><td> GST - Vip1 </td><td> 17 </td><td> 17 </td></tr><tr><td> 그의 - IP6K1 </td><td> 18 </td><td> 18 </td></tr><tr><td> 그의 - Ddp1 </td><td> 실험실에서 사용 가능 </td><td> 실험실에서 사용 가능 </td></tr><tr><td> 아크릴 아미드 : 비스 - 아크릴 아미드 19시 1분 (40 %) </td><td> Flowgen </td><td> H16972 </td></tr><tr><td> 암모늄 Persulfate (APS) </td><td> 시그마 - 알드리치 </td><td> A9164 </td></tr><tr><td> 트리스 / Borate / EDTA (에틸렌 다이아 민 테트라 초산) (TBE) </td><td> 시그마 - 알드리치 </td><td> T 9060 </td></tr><tr><td> Temed </td><td> BDH </td><td> 43083G </td></tr><tr><td> Toluidine 블루 </td><td> 시그마 - 알드리치 </td><td> 198161 </td></tr><tr><td> Speed​​Vac </td><td> 그리스도 </td><td> 100218 </td></tr><tr><td> 젤 장치 </td><td> Hoefer </td><td> SE600 </td></tr><tr><td> 진공 매니폴드 </td><td> 그리스도 </td><td> 알파 2-4 </td></tr><tr><td> 배기기 </td><td> ABM Greiffenberger </td><td> 4EKF63CX </td></tr></tbody></table>

preparation of quality inositol pyrophosphates

묘 - 이노시톨도 수정되지 않은 자연의 이상의 복잡한 phosphorylated derivates에 속해 있습니다. 최신, 진핵 세포에서 가장 풍부한 두 사람은 이노시톨 pentakisphosphate IP (5)과 이노시톨 hexakisphosphate (phytic 산성 또는 IP 6)입니다. IP 5 IP 6 하나 이상의 나트륨 채권 하나를 포함 이노시톨 나트륨 분자의 엽 성의 전구 물질입니다. IP 6 인산화은 diphoshoinositolpentakisphosphate IP (7 PP - IP 5) 및 ​​bisdiphoshoinositoltetrakisphosphate (IP 8 (PP) 2 - IP 4) 생성합니다. 이노시톨 pyrophosphates는 지금까지 공부를 모든 진핵 생물에서 고립되어있다. 또한, 이노시톨 나트륨 합성에 대한 책임 효소의 두 개의 클래스는 매우 진화 2-4 걸쳐 보존되어 있습니다. IP 6 kinases (IP 6 KS) posses 거대한 촉매 유연성, 5 IP와 PP - IP 4 각각 IP 7 IP 6 이후, 기판 이러한 제품을 사용하여 더 복잡한 분자의 생성 5,6을 촉진 변환 . 최근 나트륨 생성 효소의 두 번째 클래스는 IP 7 8 7,8 IP에 IP 6 변환할 수있는 효모 단백질 VIP 1의 양식 (도 PP - IP 5 K로 함)에서 확인되었다. 이노시톨 pyrophosphates는 인슐린 분비 9, telomere의 길이 10,11, chemotaxis 12 소낭성의 인신 매매 13, 인산 항상성 14 HIV - 1 개그 출시 15 여러 이질적인 세포 과정을 조절. 동작의 두 가지 메커니즘이 분자의 클래스에 대한 제안되었습니다. 그들은 allosterically AKT 16과 같은 특정 단백질과 상호 작용하여 세포 기능에 영향을 줄 수 있습니다. 또는 나트륨 그룹은 사전 phosphorylated 단백질 17 인산염을 기부하실 수 있습니다. 이 연구 분야의 무한한 잠재력은 이러한 분자이 새 게시물 - translational 수정을 공부하고 많은 과학자를 방해 이노시톨 pyrophosphates의 상용 소스의 부재에 의해 방해합니다. 이노시톨 pyrophosphates을 분리하기 위해 현재 사용할 수있는 방법은 복잡한 색층 장치 18,19 필요합니다. 이러한 절차는 이노시톨 나트륨 저하 20 그러므로 가난한 회복으로 이어질 수 산성 조건을 사용합니다. 또한, 성가신 포스트 컬럼 탈염 절차는 전문 실험실 그들의 사용을 제한할 수 있습니다. 본 연구에서는 우리는 IP 6 키나제와 PP - IP 5 kinases 반응의 제품의 생성, 절연 및 정화를위한 undemanding 방법을 설명합니다. 이 방법은 매우 phosphorylated 이노시톨 polyphosphates 20를 해결하기 위해 polyacrylamide 젤 전기 영동 (PAGE)의 능력에 의해 가능했다. IP 6 K1과 기판과 같은 IP 6을 사용 PP - IP 5 K 효소 반응에 따라, 페이지는 이후 물에 eluted 있던 생성 이노시톨 pyrophosphates를 구분하는 데 사용되었다.

Watch this Scientific Journal Video about Preparation of Quality Inositol Pyrophosphates at JoVE.com

Preparation of Quality Inositol Pyrophosphates

이노시톨 pyrophosphates 인간 pathologies에 중요한 역할 등 암, 당뇨병과 비만을 재생할 수 있지만, 행동의 정확한 메커니즘은 분쟁의 문제입니다. 상용 이노시톨 pyrophosphates의 부족 상세한 연구는 렌더링 문제. 여기 이노시톨 pyrophosphates의 밀리그램을 생산하고 격리하는 간단한 프로토콜을 설명합니다.

품질 이노시톨 Pyrophosphates의 준비

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

preparation-of-quality-inositol-pyrophosphates

Nanyang Technological University

Research

JoVE Journal

Biology

13.0K 조회수.  University College London. 이노시톨 pyrophosphates 인간 pathologies에 중요한 역할 등 암, 당뇨병과 비만을 재생할 수 있지만, 행동의 정확한 메커니즘은 분쟁의 문제입니다. 상용 이노시톨 pyrophosphates의 부족 상세한 연구는 렌더링 문제. 여기 이노시톨 pyrophosphates의 밀리그램을 생산하고 격리하는 간단한 프로토콜을 설명합니다.

비디오: Preparation of Quality Inositol Pyrophosphates

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

망막의 수평 슬라이스 준비

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

연구

생물학

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

의 DNA Microarrays : 샘플 품질 제어, 배열 하이브 리다이 제이션 및 스캔

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

엘루시브 작은 포유류의 고품질 DNA를 얻기 위해 비침 투 헤어 샘플링 기법

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

갑각류와 장기 치명적인 독성 시험 Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

높은 처리량 시퀀싱을위한 고품질 염색질 면역 침전의 DNA 템플릿의 생성 (칩 SEQ)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

DNA-가교 폴리 아크릴 아미드 하이드로 겔의 제조

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

거대 분자 3D 재건을위한 샘플 준비에 프라이머 높은 품질 데이터 수집 : 곳을 알아내는 - 전자 현미경의 것과하지 말아야를 수행

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

A Web Tool for Generating High Quality Machine-readable Biological Pathways

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological information. The pathways generated by this online application are machine-readable and fully compatible with essentially all web-browsers and computer operating systems. It uses a specially developed, web-enabled pathway drawing interface that permits the selection and placement of different combinations of pre-drawn biological or biochemical entities to depict reactions, interactions, transport processes and binding events. This palette of entities consists of chemical compounds, proteins, nucleic acids, cellular membranes, subcellular structures, tissues, and organs. All of the visual elements in it can be interactively adjusted and customized. Furthermore, because this tool is a web server, all pathways and pathway elements are publicly accessible. This kind of pathway &#34;crowd sourcing&#34; means that PathWhiz already contains a large and rapidly growing collection of previously drawn pathways and pathway elements. Here we describe a protocol for the quick and easy creation of new pathways and the alteration of existing pathways. To further facilitate pathway editing and creation, the tool contains replication and propagation functions. The replication function allows existing pathways to be used as templates to create or edit new pathways. The propagation function allows one to take an existing pathway and automatically propagate it across different species. Pathways created with this tool can be &#34;re-styled&#34; into different formats (KEGG-like or text-book like), colored with different backgrounds, exported to BioPAX, SBGN-ML, SBML, or PWML data exchange formats, and downloaded as PNG or SVG images. The pathways can easily be incorporated into online databases, integrated into presentations, posters or publications, or used exclusively for online visualization and exploration. This protocol has been successfully applied to generate over 2,000 pathway diagrams, which are now found in many online databases including HMDB, DrugBank, SMPDB, and ECMDB.

PathWhiz is a comprehensive, online pathway drawing tool for generating biochemical and biological pathways. It uses publicly accessible databases and easily expandable palettes consisting of pre-drawn pathway components. This protocol describes how to easily build new pathways, replicate and edit existing pathways, and propagate previously drawn pathways to different organisms.

생성 고품질 기계 판독 가능한 생물 진학을위한 웹 도구

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

조밀도 기온 변화도 원심 분리를 사용 하 여 차동 품질의 정자 컬렉션

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Field Identification of Matricaria chamomilla using a Portable qPCR System

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

Field Identification of Matricaria chamomilla using a Portable qPCR System

Presented here is a protocol for field identification of Matricaria chamomilla using a portable qPCR system. This easy-to-perform protocol is ideal as a method to confirm the identity of a botanical species at locations where access to laboratory equipment and expertise is limited, such as farms and warehouses.

휴대용 qPCR 시스템을 이용한 마이트리카리아 샤모말라의 현장 식별

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological ...