Name: preparation of quality inositol pyrophosphates
Uploaded: 2011-09-03T14:00:00
Duration: 10 min 34 s
Description: Watch this Scientific Journal Video about Preparation of Quality Inositol Pyrophosphates at JoVE.com

हम उपयोगी टिप्पणी के लिए ए Riccio धन्यवाद और पांडुलिपि पढ़ने के. यह काम चिकित्सा अनुसंधान परिषद (MRC) सेल बायोलॉजी यूनिट के लिए धन और मानव फ्रंटियर विज्ञान कार्यक्रम अनुदान (RGP0048/2009-C) द्वारा समर्थित किया गया.

1. Enzymatic रिएक्शन - 1 दिन (दोपहर में 1 घंटे) <ol><li> पहला कदम 10-20 स्वतंत्र एंजाइमी प्रतिक्रियाओं जिसमें 6 आईपी K1 या VIP1 pyrophosphorylated isoforms 6 आईपी बदलने को तैयार है . </li><li> हम उसका आईपी 6 K1 और जीएसटी - Vip1 एंजाइमों ई. से शुद्ध का उपयोग करें प्रोटोकॉल के अनुसार कोली पहले 17,18 वर्णित. </li><li> 50 μL 1X प्रतिक्रिया बफर (30 मिमी 6.8 पीएच Hepes, 50 मिमी NaCl, 6 मिमी MgSO 4, 1 मिमी डीटीटी), 6 मिमी PhosphoCreatine (पीसीआर), 25 यू / एमएल CreatinePhosphoKinase (CPK), 5 मिमी एटीपी (मिलीग्राम से युक्त प्रतिक्रियाओं तैयार नमक), 0.3 मिमी IP6, 0.05-0.1 μg उसका आईपी 6 K1 या जीएसटी Vip1. MiliQ DDH 2 ओ के साथ मात्रा समायोजित करें </li><li> संक्षेप में प्रतिक्रिया और 37 पर सेते ° सी रातोंरात रोटेशन के साथ स्पिन. </li></ol> 2. Polyacrylamide जेल कास्टिंग और लोडिंग - 2 दिन (दोपहर में 4 घंटे) <ol><li> polyacrylamide जेल 24 लंबा, 18 सेमी चौड़ा गिलास प्लेट सेमी और 1.5 मिमी चौड़े spacers का उपयोग करने के लिए तैयार है. आम तौर पर एक 16 लेन या एक प्रारंभिक सिंगल लेन कंघी इस्तेमाल किया जाता है. </li><li> 35.5% (w / v) Acrylamide:: बीआईएस Acrylamide 19:01, अमोनियम 1X Tris / / borate EDTA (TBE), 0.05% (w / v) (persulfate ए पी एस (50 एमएल / जेल) मिश्रण निम्नलिखित युक्त तैयार ), 0.05% (w / v) Temed. पूर्व casted गिलास प्लेटों के बीच मिश्रण डालो, कंघी सम्मिलित और आरटी पर 30-60 मिनट के लिए polymerize चलो. </li><li> एक बार जेल polymerized है, ठंडे कमरे में तंत्र हस्तांतरण और पूर्व 200-300 वोल्ट पर 30-60 मिनट के लिए के बारे में 1X TBE में चलाने के. </li><li> प्रत्येक प्रतिक्रिया के लिए OrangeG डाई के 1X (10 मिमी pH7.0 Tris - एचसीएल, 1 मिमी EDTA, 30% ग्लिसरॉल, 0.1% OrangeG) जोड़ें. 6 आईपी 2 nmol युक्त एक मानक नियंत्रण के रूप में लोड करने नमूना तैयार. </li><li> बफर एक सिरिंज और किसी भी वेग को निकालने के लिए एक 21G सुई का उपयोग कर चलाने के साथ प्रत्येक अच्छी तरह से अच्छी तरह धो, तो जेल लोड. पक्ष कुओं पर लोड हो रहा से बचें. </li><li> 450-550 वोल्ट (7 mAmp / जेल) में रात भर जेल चलाएँ, जब तक OrangeG डाई बैंड जेल के नीचे से पिछले 10 सेमी के भीतर है. </li></ol> 3. 7 आईपी अलगाव - 3 दिन (4 घंटे) और 4 दिन (6-7 घंटे SpeedVac सुखाने की प्रक्रिया) <ol><li> जेल तंत्र जुदा और ध्यान से एक गिलास प्लेट एक दूसरे पर जेल छोड़ने हटायें. काटें बस OrangeG डाई बैंड के ऊपर से एक जेल के एक छोटे से हिस्से आईपी 6 मानक और एक नमूना लेन युक्त नीचे करने के लिए, जैसा कि चित्र 1 में दिखाया गया है. </li><li> Toluidine ब्लू (0.1% (w / v), 20 (w / v)% मेथनॉल, toluidine नीले 2 (w / v)% ग्लिसरॉल) के साथ कुछ मिनट के लिए जेल की कटौती भाग (1-3 मिनट) या दाग inositol पाइरोफॉस्फेट बैंड तक दिखाई देता है. कांच पहले सूखने से अस्थिर जेल को रोकने के लिए जेल की शीर्ष पर वापस हटा थाली रखो. </li></ol> IP 7 बैंड दिखाई जा सकता है के बाद से यह थोड़ा IP 6 मानक की तुलना में धीमी रन चाहिए. एटीपी है, जो 6 आईपी की तुलना में तेजी से रन भी दिखाई दे सकता है (चित्रा 1 ) चाहिए. एक समाधान de धुंधला हो जाना (20 (w / v)% मेथनॉल) में कुछ मिनट के लिए जेल के दाग भाग स्थानांतरण, दूर Toluidine ब्लू के किसी भी अतिरिक्त धोने और बेदाग जेल के साथ जेल reposition. यदि उच्च pyrophosphorylated inositol isoforms (8 आईपी और आईपी 9) के दृश्य की आवश्यकता है, Toluidine ब्लू धुंधला समाधान के साथ कमरे के तापमान पर 20 मिनट के लिए जेल दाग. बाद में, दूर के बारे में 15 मिनट के लिए समाधान de धुंधला हो जाना के साथ Toluidine ब्लू धो लो. <ol start="3"><li> एक धार के साथ संदर्भ के रूप में आईपी 7 पलायन दाग जेल (चित्रा 1) के साथ निर्धारित स्थिति का उपयोग कर जेल की बेदाग भाग पर आईपी 7 बैंड में कटौती. </li><li> आईपी ​​7 बैंड है कि जेल से एक 15 एमएल ट्यूब पर काट दिया गया रखो और MilliQ DDH 2 ओ के 10 एमएल जोड़ने रोटेशन में कमरे के तापमान पर 10 मिनट के लिए ट्यूबों का रखो. TBE और सूक्ष्म acrylamide कणों से अधिक दूर तरल त्यागें. </li><li> बाद में, दो निर्जलीकरण जलयोजन चक्र करते हैं. ट्यूब आईपी 7 युक्त जेल के साथ 50 (w / v)% मेथनॉल के 5 एमएल जोड़ें 2 घंटे के लिए कमरे के तापमान पर बारी बारी से. एक नया 15 एमएल MilliQ DDH 2 हे 5 एमएल युक्त ट्यूब जेल टुकड़ा स्थानांतरण और 2 घंटे के लिए कमरे के तापमान पर बारी बारी से. और ट्यूब से मेथनॉल MilliQ एच 2 हे त्यागने मत करो. निर्जलीकरण - जलयोजन चक्र फिर 15 एमएल पहले प्रयोग किया जाता ट्यूबों स्थानांतरित में polyacrylamide जेल से एक बार और दोहराएँ. Washes के एक रात खत्म किया जा सकता है. </li><li> Eluted 7 आईपी ध्यान केंद्रित करने के लिए, 10 एमएल के साथ शुष्क (5 एमएल MilliQddH 2 हे और 5 एमएल 50% मेथनॉल (w / v)) एक 60 में गर्म SpeedVac ° सी. का उपयोग </li><li> एक बार नमूने लगभग सूख रहे हैं, a1.5 एमएल अपकेंद्रित्र ट्यूब और 5000 rpm पर 2 मिनट के लिए स्पिन करने के लिए शेष तरल हस्तांतरण (300-600 μl). </li><li> एक ताजा 1.5 एमएल अपकेंद्रित्र ट्यूब में सतह पर तैरनेवाला और हस्तांतरण लीजिए; छोड़नीचे 20-30 μL के बाद से यह acrylamide कणों को शामिल कर सकते हैं. </li><li> यदि आवश्यक हो सुखाने प्रक्रिया का उपयोग कर एक unheated SpeedVac जारी है. 7 आईपी वसूली को नाटकीय रूप से अगर नमूने पूरी तरह से सूखे कम है, इसलिए सुखाने की प्रक्रिया समाप्त जब नमूने 100-300 μL की मात्रा तक पहुँचने. </li></ol> 4. आईपी ​​7 एकाग्रता और पवित्रता का निर्धारण. <ol><li> बरामद आईपी 7 नमूना एक पन्ना जेल पर चलाने के 2-5 μL इसी तरह 2.2-2.5 अनुभाग का उपयोग करें . एकाग्रता मानक और पाली - पी मार्कर के 4 nmol 6 आईपी कई dilutions (यानी 0.5, 1, 2, 4 nmol) के रूप में लोड करें. जेल चलाने के बाद, धुंधला हो जाना और धुंधला हो जाना Toluidine ब्लू समाधान के साथ पूरे जेल द्वारा inositol पाइरोफॉस्फेट isoforms 3.2 धारा (चित्रा 2A) में वर्णित प्रक्रिया के बाद, कल्पना. </li><li> Toluidine धुंधला के बाद, सांद्रता जेल स्कैनिंग और तीव्रता में 6 आईपी और आईपी 7 के बीच मतभेद की तुलना, जैसे छवि जम्मू इमेजिंग सॉफ्टवेयर का उपयोग कर के रूप में चित्रा 2B में दिखाया गया है द्वारा निर्धारित किया जा सकता है. </li></ol> 5. प्रतिनिधि परिणाम: 6 आईपी प्रारंभिक एंजाइमी 7 आईपी आईपी 6 K1 और VIP1 एंजाइमों का उपयोग करने के लिए रूपांतरण PAGE विश्लेषण (चित्रा 1) का उपयोग आसानी से हल कर सकते हैं. लोड हो रहा है की एक आकार पर नियंत्रण के रूप में आईपी Toluidine ब्लू जेल धुंधला के साथ एक साथ 6 pyrophosphorylated derivates की पहचान की अनुमति देता है, क्योंकि वे धीमी चलाने फॉस्फेट inositol अंगूठी पर मौजूद समूहों की संख्या पर निर्भर करता है. 7 आईपी के ऊपर वर्णित की प्रक्रिया आसान शुद्धि की अनुमति देता है . शुद्ध पन्ना पाइरोफॉस्फेट inositol का विश्लेषण हमारे 7 आईपी (2A चित्रा) की शुद्धता का पता चला. दिलचस्प है, VIP1 के आईपी 7 उत्पाद के 1/3PP-IP 5 isomer थोड़ा 5PP आईपी 7 आईपी के 5 isomer है कि आईपी 6 K1 द्वारा उत्पन्न होता है की तुलना में धीमी migrates . आईपी ​​6 मानकों का प्रयोग शुद्ध 7 आईपी (चित्रा 2B) की एकाग्रता का एक आसान मात्रा का ठहराव की अनुमति. आगे के प्रयोगों के लिए आईपी 7 का उपयोग करने से पहले, अपने जैविक गतिविधि का मूल्यांकन किया जा सकता है (चित्रा 3). 5PP आईपी 5 VIP1 के साथ और आईपी 7 DDP1 फॉस्फेट (diphosphoinositol polyphosphate phosphohydrolase) के साथ incubated है . नियमित, शुद्ध 7 आईपी DDP1 (चित्रा 3) में VIP1 द्वारा 8 आईपी और आईपी 6 कनवर्ट किया जाता है . <img alt="चित्रा 1" src="/files/ftp_upload/3027/3027fig1.jpg" /> चित्रा 1: PAGE और आईपी 7 बैंड के अलगाव के Toluidine धुंधला मानक (6 आईपी) युक्त जेल के हिस्से में कटौती करने के लिए और एक Toluidine ब्लू समाधान का उपयोग दाग. तीन बैंड (ऊपर से नीचे) 7 आईपी, 6 आईपी और एटीपी प्रतिनिधित्व करते हैं. जेल के दाग भाग तो जेल के शेष के साथ गठबंधन किया गया था. यह आईपी 7, जो तब और कटौती जा सकता है शुद्ध (डैश्ड बॉक्स) से युक्त जेल के हिस्से का स्थानीयकरण अनुमति देता है . <img alt="चित्रा 2" src="/files/ftp_upload/3027/3027fig2.jpg" /> चित्रा 2: आईपी 6 K1 और VIP1 प्रतिक्रिया उत्पादों के PAGE विश्लेषण. ए) 6 आईपी Toluidine ब्लू धुंधला द्वारा विश्लेषण (4, 2, 1, 0.5 nmol) क्रम में आईपी 7 से दोनों आईपी 6 K1 (5 5PP - आईपी) और VIP1 (1/3PP-IP शुद्ध एकाग्रता का निर्धारण करने के लिए इस्तेमाल किया गया था ) 5 प्रतिक्रियाओं.) बी स्कैटर साजिश विश्लेषण शुद्ध 7 आईपी की एकाग्रता का निर्धारण करने के लिए. सांद्रता बैंड तीव्रता, imageJ सॉफ्टवेयर का उपयोग कर की गणना 6 आईपी के पूर्व निर्धारित मात्रा की तुलना में, अनुसार निर्धारित किया गया है. X-अक्ष तीव्रता का प्रतिनिधित्व करता है, Y-अक्ष nmol में व्यक्त की सांद्रता का प्रतिनिधित्व करता है. <img alt="चित्रा 3" src="/files/ftp_upload/3027/3027fig3.jpg" /> चित्रा 3: आईपी 7 जैविक गतिविधि का विश्लेषण शुद्ध 7 आईपी की गुणवत्ता निर्धारित करने के लिए हम VIP1 या आईपी 7 फॉस्फेट के साथ DDP1 5 5PP आईपी (आईपी 6 K1 उत्पन्न आईपी 7) incubated और तब पृष्ठ पर प्रतिक्रिया हल. DAPI और Toluidine धुंधला VIP1 द्वारा 8 आईपी की उम्मीद उत्पादन और 7 आईपी DDP1 द्वारा 6 आईपी रूपांतरण का पता चला.

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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>

ब्याज की कोई संघर्ष की घोषणा की.

जैव रसायन में inositol पाइरोफॉस्फेट का उपयोग गंभीर रूप से ऐसे यौगिकों के वाणिज्यिक अनुपलब्धता और मौजूदा तरीकों का पता लगाने के गरीब संवेदनशीलता द्वारा सीमित है. पन्ना है, जो फॉस्फेट समूहों के विभिन्न संख्या है, और Toluidine (चित्र 1) ब्लू, एक metachromatic डाई जो फॉस्फेट समूहों को बांधता है, रखने के अणुओं की जुदाई में सक्षम बनाता है के संयोजन inositol pyrophoshate 20 अनुसंधान के नए रास्ते खोलने isoforms का आसान पता लगाने के लिए सक्षम बनाता है. पृष्ठ प्रौद्योगिकी के वर्णित उपयोग enzymatic प्रतिक्रिया के inositol पाइरोफॉस्फेट उत्पादों शुद्ध outby किया जाता है या तो आईपी K1 6 या VIP1 एक सरल, आर्थिक और विश्वसनीय तरीका है कि उच्च गुणवत्ता 7 आईपी की बड़ी मात्रा में के उत्पादन के लिए अनुमति देता है. ऊपर वर्णित विधि आईपी के सरल शुद्धि के लिए 7 सीमित नहीं है, लेकिन वर्णित प्रोटोकॉल के मामूली संशोधनों inositol pyrophosphates का एक अलग श्रेणी की शुद्धि की अनुमति दे सकता है. हायर phosphorylated inositol पाइरोफॉस्फेट isoforms, आठ से अधिक फॉस्फेट समूहों से युक्त एक 20,6 सब्सट्रेट के रूप में 7 आईपी या आईपी 6 के विभिन्न मात्रा का उपयोग कर पता लगाया जा सकता है. ये inositol pyrophosphates धुंधला प्रक्रिया की लंबाई बढ़ती है और बाद में शुद्ध (3.2 अनुभाग) के द्वारा पता लगाया जा सकता है. इसके अलावा, enzymatic प्रतिक्रिया के लिए सब्सट्रेट के रूप में 5 आईपी का उपयोग 5 पीपी आईपी की शुद्धि और अन्य inositol inositol की अंगूठी पर एक हाइड्रॉक्सिल समूह युक्त pyrophosphates की अनुमति होगी. अंत में, इस उदार विधि व्यापक रूप से उपलब्ध उपकरणों के साथ inositol pyrophosphates milligram मात्रा के विश्वसनीय शुद्धि के लिए अनुमति देता है, इस प्रकार इस रोमांचक अनुसंधान के क्षेत्र के लिए नए रास्ते खोलने.

<table border="1"><tbody><tr><th> अभिकर्मक के नाम </th><th> कंपनी </th><th> सूचीपत्र संख्या </th></tr><tr><td> Phytic एसिड (6 आईपी) </td><td> सिग्मा Aldrich </td><td> P8810 </td></tr><tr><td> पाली - पी (सोडियम hexametaphosphate) </td><td> सिग्मा Aldrich </td><td> P8510 </td></tr><tr><td> एटीपी मिलीग्राम 2 + नमक </td><td> सिग्मा Aldrich </td><td> A9187 </td></tr><tr><td> OrangeG </td><td> सिग्मा Aldrich </td><td> O3756 </td></tr><tr><td> PhosphoCreatine (पीसीआर) </td><td> सिग्मा Aldrich </td><td> P7936 </td></tr><tr><td> CreatinePhospho kinase (CPK) </td><td> सिग्मा Aldrich </td><td> C3755 </td></tr><tr><td> जीएसटी के Vip1 </td><td> 17 </td><td> 17 </td></tr><tr><td> उनकी-IP6K1 </td><td> 18 </td><td> 18 </td></tr><tr><td> उनकी - Ddp1 </td><td> प्रयोगशाला में उपलब्ध </td><td> प्रयोगशाला में उपलब्ध </td></tr><tr><td> Acrylamide: 19:01 बीआईएस Acrylamide (40%) </td><td> Flowgen </td><td> H16972 </td></tr><tr><td> अमोनियम Persulfate (ए पी) </td><td> सिग्मा Aldrich </td><td> A9164 </td></tr><tr><td> Tris / / borate EDTA (TBE) </td><td> सिग्मा Aldrich </td><td> 9060 टी </td></tr><tr><td> Temed </td><td> BDH </td><td> 43083G </td></tr><tr><td> Toluidine ब्लू </td><td> सिग्मा Aldrich </td><td> 198161 </td></tr><tr><td> SpeedVac </td><td> मसीह </td><td> 100218 </td></tr><tr><td> जेल तंत्र </td><td> Hoefer </td><td> SE600 </td></tr><tr><td> वैक्यूम कई गुना </td><td> मसीह </td><td> 2-4 अल्फा </td></tr><tr><td> वेक्यूम - पंप </td><td> एबीएम Greiffenberger </td><td> 4EKF63CX </td></tr></tbody></table>

preparation of quality inositol pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

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

Preparation of Quality Inositol Pyrophosphates

Inositol pyrophosphates मानव विकृतियों में एक महत्वपूर्ण भूमिका निभाते हैं जैसे कैंसर, मधुमेह और मोटापा, तथापि, कार्रवाई की सटीक व्यवस्था विवाद का एक मामला है. व्यावसायिक रूप से उपलब्ध inositol pyrophosphates की कमी renders विस्तृत अध्ययन समस्याग्रस्त. यहाँ हम एक सरल उत्पादन और inositol pyrophosphates के मिलीग्राम अलग प्रोटोकॉल का वर्णन.

गुणवत्ता Inositol Pyrophosphates की तैयारी

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

Research

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Biology

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.

नमूना गुणवत्ता नियंत्रण, ऐरे संकरण और स्कैन: डीएनए

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.

एक अवेध्य बालों नमूनाकरण मायावी छोटे स्तनधारी से उच्च गुणवत्ता डीएनए प्राप्त तकनीक

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.

Crustacean साथ लंबी अवधि के घातक विषाक्तता टेस्ट Artemia franciscana</em

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

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.

Do&#039;s and Don&#039;ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

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.

MACROMOLECULAR 3D पुनर्निर्माण के लिए एक नमूना तैयार करने पर प्राइमर और उच्च गुणवत्ता वाले डेटा संग्रह: क्रायो-इलेक्ट्रॉन माइक्रोस्कोपी की है और don&#39;ts

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.

जनरेटिंग उच्च गुणवत्ता की मशीन पठनीय जैविक रास्ते के लिए एक वेब उपकरण

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.

शुक्राणु का संग्रह विभेदक गुणवत्ता घनत्व ढाल केंद्रापसारक का उपयोग

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.

पोर्टेबल क्यूपीसीआर सिस्टम का उपयोग करके मैट्रिकिया चामोमिला की फील्ड पहचान

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

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

रूट एक्सूडेट्स में Rhizobacterial Chemoattractants की तेजी से पहचान के लिए एक बेहतर Chemotaxis परख

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as yogurt and cheese. LAB predominantly produce lactic acid as a major end product of fermentation, and they synthesize important metabolites that impart the organoleptic characteristics of fermented food products. LAB are fastidious bacteria that thrive in many environments when adequate nutritional requirements are fulfilled. The demand for superior LAB dairy starter cultures for fermentation applications in the food and dairy industry, has resulted in the need to provide viable and active cultures for all bioprocessing operations. The development of a standard protocol for ensuring the viability and enhanced functionality of LAB cultures in the laboratory as well as dairy processing environments is thus very critical. In addressing concerns linked to resuscitating weak, stressed, and injured LAB culture cells, a protocol that vividly outlines salient steps to recover, enhance cell regeneration, and improve metabolic functionality of LAB strains is of the utmost importance. The maintenance of culture purity, functionality, and viability for LAB starter cultures is likewise critical. Therefore, adherence to a unique protocol guideline will result in the promotion of fermentation performance for many LAB strains dedicated to fermentation and biotechnology processes. As a result, the Food Microbiology and Biotechnology Laboratory at North Carolina Agriculture and Technical State University has developed a standard protocol for the activation and quality control of selected LAB strains that has resulted in highly functional and viable LAB culture strains employed for fermentation research. The adaptation and recommendation of a protocol such as this for use in the dairy and food industry&#160;will help to ensure LAB viability and functionality for many applications.

The quality control assessment of lactic acid bacteria (LAB) cultures has been confirmed as an effective way to enhance the viability and functionality of LAB strains for fermentation procedures. To buttress this assertion, we developed a protocol that elucidates how LAB cultures are activated and cultivated for fermentation and bioprocessing procedures.

लैक्टिक एसिड बैक्टीरिया की खेती, विकास और व्यवहार्यता: एक गुणवत्ता नियंत्रण परिप्रेक्ष्य

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as ...

प्रक्रिया विकास के माध्यम से स्प्रे-सूखे प्रोबायोटिक उत्पाद की गुणवत्ता का अनुकूलन

Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that can confer beneficial effects on human and animal wellbeing, while prebiotics are types of nutrients that feed the beneficial gut bacteria. Powder probiotics have gained popularity due to the ease and practicality of their ingestion and incorporation into the diet as a food supplement. However, the drying process interferes with cell viability since high temperatures inactivate probiotic bacteria. In this context, this study aimed to present all the steps involved in the production and physicochemical characterization of a spray-dried probiotic and evaluate the influence of the protectants (simulated skim milk and inulin:maltodextrin association) and drying temperatures in increasing the powder yield and cell viability. The results showed that the simulated skim milk promoted higher probiotic viability at 80 &#176;C. With this protectant, the probiotic viability, moisture content, and water activity (Aw) reduce as long as the inlet temperature increases. The probiotics&#39; viability decreases conversely with the drying temperature.&#160;At temperatures close to 120 &#176;C, the dried probiotic showed viability around 90%, a moisture content of 4.6% w/w, and an Aw of 0.26; values adequate to guarantee product stability. In this context, spray-drying temperatures above 120 &#176;C are required to ensure the microbial cells&#39; viability and shelf-life in the powdered preparation and survival during food processing and storage.

लेखक स्पॉटलाइट: प्रोबायोटिक बैक्टीरिया के स्प्रे-सुखाने और उत्पाद की गुणवत्ता के मूल्यांकन के लिए प्रक्रिया विकास  

This protocol details the steps involved in the production and physicochemical characterization of a spray-dried probiotic product.

प्रोबायोटिक बैक्टीरिया के स्प्रे-सुखाने और उत्पाद की गुणवत्ता के मूल्यांकन के लिए प्रक्रिया विकास

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that ...