Name: lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (limacs): a novel method to analyze protein-lipid interaction
Uploaded: 2011-04-26T16:00:00
Description: Watch this Scientific Journal Video about Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction at JoVE.com

इस काम एनआईएच R01NS046835 अनुदान और R01AG034389, और मार्च ऑफ डाइम्स 6FY08 322 अनुदान के द्वारा समर्थित किया गया था. एक विशेष धन्यवाद आप श्रीमती Eleanor (Miltenyi बायोटेक, Auburn, CA) भूरी जो उसके अंतर्दृष्टि के साथ काफी एमएसीएस प्रौद्योगिकी में मदद करने के लिए समर्पित है. Miltenyi उदारता के प्रयोगों के प्रदर्शन के लिए कोई भी कीमत पर सामग्री का इस्तेमाल किया प्रदान की है. मैं भी डॉ. Guanghu वैंग (/ जॉर्जिया जॉर्जिया स्वास्थ्य विज्ञान विश्वविद्यालय, Augusta के मेडिकल कॉलेज, GA) जो सेल लाइनों को व्यक्त PKCζ उत्पन्न करने के लिए आभारी हूँ. जॉर्जिया / जॉर्जिया स्वास्थ्य विज्ञान विश्वविद्यालय (डॉ. लिन मेई के निदेशक के तहत) के मेडिकल कॉलेज में आण्विक चिकित्सा के संस्थान द्वारा समर्थन भी स्वीकार किया है.

1. परिचय लिपिड पुटिका की मध्यस्थता आत्मीयता क्रोमैटोग्राफी चुंबकीय सक्रिय कक्ष (LIMACS) छँटाई तकनीक का उपयोग कर हमारी प्रयोगशाला में विकसित किया गया था करने के लिए ceramide जुड़े प्रोटीन परिसरों को...

<ol>
	<li>Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+binding+to+ceramide+activates+protein+kinase+Czeta+before+the+formation+of+a+pro-apoptotic+complex+with+PAR-4+in+differentiating+stem+cells.">Direct binding to ceramide activates protein kinase Czeta before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells.</a> J Biol Chem. 280, 26415-26424 (2005).</li><li>Wang, G., Krishnamurthy, K., Umapathy, N. S., Verin, A. D., Bieberich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+carboxyl-terminal+domain+of+atypical+protein+kinase+Czeta+binds+to+ceramide+and+regulates+junction+formation+in+epithelial+cells.">The carboxyl-terminal domain of atypical protein kinase Czeta binds to ceramide and regulates junction formation in epithelial cells.</a> J Biol Chem. 284, 14469-14475 (2008).</li><li>Chalfant, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+ceramide+regulates+the+alternative+splicing+of+caspase+9+and+Bcl-x+in+A549+lung+adenocarcinoma+cells.+Dependence+on+protein+phosphatase-1.">De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1.</a> J Biol Chem. 277, 12587-12595 (2002).</li><li>Simon, C. G., Holloway, P. W., Gear, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exchange+of+C(16)-ceramide+between+phospholipid+vesicles.">Exchange of C(16)-ceramide between phospholipid vesicles.</a> Biochemistry. 38, 14676-14682 (1999).</li><li>Goni, F. M., Contreras, F. X., Montes, L. R., Sot, J., Alonso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysics+(and+sociology)+of+ceramides.">Biophysics (and sociology) of ceramides.</a> Biochem Soc Symp. , 177-188 (2005).</li><li>Kumagai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CERT+mediates+intermembrane+transfer+of+various+molecular+species+of+ceramides.">CERT mediates intermembrane transfer of various molecular species of ceramides.</a> J Biol Chem. 280, 6488-6495 (2005).</li><li>Wessel, D., Flugge, U. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+quantitative+recovery+of+protein+in+dilute+solution+in+the+presence+of+detergents+and+lipids.">A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.</a> Anal Biochem. 138, 141-143 (1984).</li></ol>

कोशिका झिल्ली में lipids की embedding द्वारा एक लिपिड और उसके बंधनकारी प्रोटीन के बीच विशिष्ट बातचीत का परीक्षण करने के लिए आड़े आती है. कोशिका झिल्ली कई lipids और प्रोटीन का एक मिश्रण के होते हैं और यह लिपिड microdomains या ra...

Annexin वि संयुग्मित चुंबकीय मोतियों और एमएसीएस सूक्ष्म और minicolumns Miltenyi बायोटेक इंक, (Auburn, सीए) द्वारा प्रदान किया गया. सभी लिपिड उच्चतम शुद्धता के थे और अवंती ध्रुवीय Lipids (सिलखड़ी, अल) से प्राप्त की.

lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (limacs): a novel method to analyze protein-lipid interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Watch this Scientific Journal Video about Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction at JoVE.com

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

अपने लक्ष्य लिपिड हम एमएसीएस और Annexin वि संयुग्मित चुंबकीय मोतियों और लिपिड लक्ष्य लिपिड और Annexin वि बाध्यकारी phosphatidylserine से संश्लेषित vesicles उपयोग के साथ एक प्रोटीन की बातचीत का परीक्षण. लक्ष्य लिपिड करने के लिए बाध्य प्रोटीन सह शुद्ध कर रहे हैं और मोती से elution बाद विश्लेषण.

लिपिड आत्मीयता पुटिका की मध्यस्थता चुंबकीय सक्रिय सेल छंटनी (LIMACS) का उपयोग कर क्रोमैटोग्राफी: प्रोटीन लिपिड इंटरेक्शन विश्लेषण के लिए एक उपन्यास विधि

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

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Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

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