Name: quantitative phosphoproteomics in fatty acid stimulated saccharomyces cerevisiae
Uploaded: 2009-10-12T23:00:00
Description: Watch this Scientific Journal Video about Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae at JoVE.com

हम तकनीकी सहायता के लिए मास स्पेक्ट्रोमेट्री विश्लेषण और उपयोगी चर्चा के साथ डा. रिच रोजर्स का शुक्रिया अदा करना होगा, और डीआरएस. रोब. Moritz, जेफ Ranish, और उपयोगी विचार - विमर्श के लिए हामिद Mirzai. इस काम उत्कृष्टता के NIH केंद्र द्वारा वित्त पोषित किया गया था.

सेल विकास और मीडिया <ol><li> एक 1.0 एक न्यूनतम खमीर मध्यम के दो 1 लीटर संस्कृतियों (0.17% अमोनियम सल्फेट या अमीनो एसिड के बिना खमीर नाइट्रोजन बेस, 0.5% अमोनियम सल्फेट) में बीज तो BY4742Δarg4Δlys1 100 एक 600 आयुध डिपो एमएल अम?...

<ol>
	<li>Albuquerque, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multidimensional+chromatography+technology+for+in-depth+phosphoproteome+analysis.">A multidimensional chromatography technology for in-depth phosphoproteome analysis.</a> Mol Cell Proteomics. 7 (7), 1389-1396 (2008).</li><li>McNulty, D. E., Annan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophilic+interaction+chromatography+reduces+the+complexity+of+the+phosphoproteome+and+improves+global+phosphopeptide+isolation+and+detection.">Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.</a> Mol Cell Proteomics. 7 (5), 971-980 (2008).</li><li>Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+complex+protein+mixtures+using+isotope-coded+affinity+tags.">Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.</a> Nat Biotechnol. 17 (10), 994-999 (1999).</li><li>Ross, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+protein+quantitation+in+Saccharomyces+cerevisiae+using+amine-reactive+isobaric+tagging+reagents.">Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.</a> Mol Cell Proteomics. 3 (12), 1154-1169 (2004).</li><li>Gruhler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phosphoproteomics+applied+to+the+yeast+pheromone+signaling+pathway.">Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.</a> Mol Cell Proteomics. 4 (3), 310-327 (2005).</li><li>Wilson-Grady, J. T., Villen, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteome+analysis+of+fission+yeast.">Phosphoproteome analysis of fission yeast.</a> J Proteome Res. 7 (3), 1088-1097 (2008).</li><li>Villen, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SCX/IMAC+enrichment+approach+for+global+phosphorylation+analysis+by+mass+spectrometry.">The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry.</a> Nat Protoc. 3 (10), 1630-1638 (2008).</li><li>Jensen, S. S., Larsen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+impact+of+some+experimental+procedures+on+different+phosphopeptide+enrichment+techniques.">Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.</a> Rapid Commun Mass Spectrom. 21 (22), 3635-3645 (2007).</li><li>Larsen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+selective+enrichment+of+phosphorylated+peptides+from+peptide+mixtures+using+titanium+dioxide+microcolumns.">Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.</a> Mol Cell Proteomics. 4 (7), 873-886 (2005).</li><li>Pinkse, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+isolation+at+the+femtomole+level+of+phosphopeptides+from+proteolytic+digests+using+2D-NanoLC-ESI-MS/MS+and+titanium+oxide+precolumns.">Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns.</a> Anal Chem. 76 (14), 3935-3943 (2004).</li><li>Tao, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phosphoproteome+analysis+using+a+dendrimer+conjugation+chemistry+and+tandem+mass+spectrometry.">Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry.</a> Nat Methods. 2 (8), 591-598 (2005).</li><li>Zhou, H., Watts, J. 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इस विधि phosphopeptides कि मात्रात्मक विश्लेषण किया जा सकता है की एक संवर्धन निकलेगा. मानकों की एक संख्या इस प्रोटोकॉल में बदला जा सकता है, लेकिन सबसे महत्वपूर्ण पहलू को याद करने के लिए अपने phosphorylation संरक्षित है. मा?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Isotec&trade; L-Arginine-13C6,15N4</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>608033</td>
<td>&nbsp;</td>
<tr>
<td>Isotec&trade; L-Lysine-15N2</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>609021</td>
<td>&nbsp;</td>
<tr>
<td>GIBCO&#x2122; Phosphate-Buffered Saline (PBS) 7.4 (10X) liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>70011044</td>
<td>&nbsp;</td>
<tr>
<td>SigmaFAST Protease Inhibitor Tablets</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8820</td>
<td>&nbsp;</td>
<tr>
<td>Pierce Halt Phosphatase Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>78420</td>
<td>&nbsp;</td>
<tr>
<td>Retsch PM100 Ball Mill Grinder </td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>20.540.0001</td>
<td>&nbsp;</td>
<tr>
<td>Retsch 125 ml Stainless Steel Grinding Jar</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>01.462.0148</td>
<td>&nbsp;</td>
<tr>
<td>Ultramicrospin C18 column</td>
<td>&nbsp;</td>
<td>The Nest Group</td>
<td>SUM SS10</td>
<td>&nbsp;</td>
<tr>
<td>Sep-Pak Vac 500 mg C18</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT043395</td>
<td>&nbsp;</td>
<tr>
<td>TSK-Gel Amide-80 4.6 mm x 25 cm analytical column</td>
<td>&nbsp;</td>
<td>TOSOH BioSciences</td>
<td>13071</td>
<td>This is the HILIC chromatographic column.</td>
</tr>
<tr>
<td>Guard column 4.6 mm ID x1 cm</td>
<td>&nbsp;</td>
<td>TOSOH BioSciences</td>
<td>19021</td>
<td>We recommend this guard column to extend the life of your HILIC column.</td>
</tr>
<tr>
<td>PHOS-Select&trade; Iron Affinity Gel</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P9740</td>
<td>This is the IMAC resin. Aliquot and store.</td>
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quantitative phosphoproteomics in fatty acid stimulated saccharomyces cerevisiae

इस प्रोटोकॉल isotopically भारी और प्रकाश एस cerevisiae कोशिकाओं के फैटी एसिड oleate, के साथ विकास और उत्तेजना का वर्णन करता है. कक्ष एक गेंद मिल चक्की और जिसके परिणामस्वरूप यूरिया solubilization द्वारा समाधान में लाया grindate में एक cryolysis प्रक्रिया का उपयोग कर भूमि रहे हैं. यह प्रक्रिया एक metabolically निष्क्रिय स्थिति में कोशिकाओं के lysis के लिए अनुमति देता है, phosphorylation के संरक्षण और सेल lysis के दौरान phosphoproteome के पुनरभिविन्यास को रोकने. कमी, alkylation, trypsin प्रोटीन के पाचन के बाद, नमूने C18 स्तंभों और नमूना हाइड्रोफिलिक बातचीत क्रोमैटोग्राफी (HILIC) का उपयोग fractionation द्वारा कम जटिलता पर desalted हैं. HILIC स्तंभों preferentially हाइड्रोफिलिक अणुओं है जो अच्छी तरह से phosphoproteomics के लिए अनुकूल है बनाए रखने. Phosphorylated पेप्टाइड्स के बाद गैर phosphorylated समकक्षों की तुलना में chromatographic प्रोफ़ाइल में elute करते हैं. Fractionation के बाद, phosphopeptides immobilized धातु क्रोमैटोग्राफी, जो phosphopeptide संवर्धन के लिए प्रभारी आधारित समानताएं पर निर्भर करता है का उपयोग कर समृद्ध कर रहे हैं. इस प्रक्रिया के अंत में नमूने के मात्रात्मक मास स्पेक्ट्रोमेट्री द्वारा विश्लेषण किया जा करने के लिए तैयार हैं.

Watch this Scientific Journal Video about Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae at JoVE.com

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

एक मात्रात्मक phosphorylation cryolysis, यूरिया solubilziation, HILIC fractionation और phosphorylated पेप्टाइड्स के iMac संवर्धन का उपयोग प्रक्रिया का विवरण.

फैटी एसिड में मात्रात्मक Phosphoproteomics प्रेरित Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

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Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Dissection of Saccharomyces Cerevisiae Asci

के विच्छेदन Saccharomyces cerevisiae एएससीआई

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

मात्रात्मक प्रोटिओमिक्स के साथ एस cerevisiae में प्रोटीन परिसरों की पहचान

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Microarray Analysis for Saccharomyces cerevisiae

Microarray Analysis for Saccharomyces cerevisiae

माइक्रोएरे विश्लेषण के लिए Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Quantitation और हो - endonuclease के गठन के विश्लेषण में एनीलिंग एकल Strand द्वारा गुणसूत्र translocations प्रेरित Saccharomyces cerevisiae</em

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

प्रतिदीप्ति का प्रयोग mRNA अणुओं के दृश्य और विश्लेषण<em&gt; स्वस्थानी</emमें&gt; संकरण<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

में निगरानी प्रोटीन refolding के लिए युग्मित Assays<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

MRNA के क्षय दरों की माप में<em&gt; Saccharomyces cerevisiae</em&gt; का प्रयोग<em&gt; Rpb1-1</em&gt; खींच

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

में रासायनिक आनुवंशिक बातचीत की तेजी से पहचान<em&gt; Saccharomyces cerevisiae</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

माउस प्राथमिक हेपाटोसाइट्स में फैटी एसिड ऑक्सीकरण और lipogenesis का निर्धारण

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

में प्रोटीन गिरावट के cycloheximide चेस विश्लेषण<em&gt; Saccharomyces cerevisiae</em

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein ...