Name: density gradient ultracentrifugation for investigating endocytic recycling in mammalian cells
Uploaded: 2021-06-30T13:15:00
Description: Watch this Scientific Journal Video about Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells at JoVE.com

Dette arbejde blev støttet af midler fra Research Grants Council Hong Kong, CUHK direkte tilskudsordning, United College begavelse fond, og TUYF Charitable Trust. Tallene i dette arbejde er tilpasset fra vores tidligere publikation, "ARF6-Rac1 signalmedieret neurit udvækst er potentiated af neuronal adapter FE65 gennem orkestrering ARF6 og ELMO1" offentliggjort i FASEB Journal i oktober 2020.

1. Pattedyrs cellekultur og transfekt
<ol><li>Plade 2 x 106 celler i en 100 mm kulturret. Brug fire retter til hver transfekt. BEMÆRK: Antallet af celler, der kræves, kan variere for forskellige cellelinjer. Optimering kan være nødvendig, før du går videre til isolationstrinnet.</li>
	<li>Den næste dag, transfect cellerne med Lipofectamin i henhold til producentens anvisninger.</li>
</ol>2. Cellehøst
<ol><li>Kassér kulturmediet 48 timer efter transfection.</li>
	<li>Cell...

<ol>
	<li>Elkin, S. R., Lakoduk, A. M., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytic+pathways+and+endosomal+trafficking:+a+primer.">Endocytic pathways and endosomal trafficking: a primer.</a> Wiener Medizinische Wochenschrift. 166 (7-8), 196-204 (2016).</li><li>Kumari, S., Mg, S., Mayor, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+unplugged:+multiple+ways+to+enter+the+cell.">Endocytosis unplugged: multiple ways to enter the cell.</a> Cell Research. 20 (3), 256-275 (2010).</li><li>Naslavsky, N., Caplan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enigmatic+endosome+-+sorting+the+ins+and+outs+of+endocytic+trafficking.">The enigmatic endosome - sorting the ins and outs of endocytic trafficking.</a> Journal of Cell Science. 131 (13), (2018).</li><li>Cullen, P. J., Steinberg, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+degrade+or+not+to+degrade:+mechanisms+and+significance+of+endocytic+recycling.">To degrade or not to degrade: mechanisms and significance of endocytic recycling.</a> Nature Reviews. Molecular Cell Biology. 19, 679-696 (2018).</li><li>Weeratunga, S., Paul, B., Collins, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognising+the+signals+for+endosomal+trafficking.">Recognising the signals for endosomal trafficking.</a> Current Opinion in Cell Biology. 65, 17-27 (2020).</li><li>Khan, I., Steeg, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis:+a+pivotal+pathway+for+regulating+metastasis.">Endocytosis: a pivotal pathway for regulating metastasis.</a> British Journal of Cancer. 124 (1), 66-75 (2021).</li><li>Grant, B. D., Donaldson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+and+mechanisms+of+endocytic+recycling.">Pathways and mechanisms of endocytic recycling.</a> Nature Reviews. Molecular Cell Biology. 10 (9), 597-608 (2009).</li><li>Maxfield, F. R., McGraw, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytic+recycling.">Endocytic recycling.</a> Nature Reviews. Molecular Cell Biology. 5 (2), 121-132 (2004).</li><li>McDonald, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explosion+in+the+complexity+of+membrane+protein+recycling.">Explosion in the complexity of membrane protein recycling.</a> American Journal of Physiology. Cell Physiology. 320 (4), 483-494 (2021).</li><li>O'Sullivan, M. J., Lindsay, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Endosomal+Recycling+pathway-at+the+crossroads+of+the+cell.">The Endosomal Recycling pathway-at the crossroads of the cell.</a> International Journal of Molecular Sciences. 21 (17), 6074 (2020).</li><li>D'Souza-Schorey, C., Li, G., Colombo, M. I., Stahl, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regulatory+role+for+ARF6+in+receptor-mediated+endocytosis.">A regulatory role for ARF6 in receptor-mediated endocytosis.</a> Science. 267 (5201), 1175-1178 (1995).</li><li>Schweitzer, J. K., Sedgwick, A. E., D'Souza-Schorey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARF6-mediated+endocytic+recycling+impacts+cell+movement,+cell+division+and+lipid+homeostasis.">ARF6-mediated endocytic recycling impacts cell movement, cell division and lipid homeostasis.</a> Seminars in Cell and Developmental Biology. 22 (1), 39-47 (2011).</li><li>Finicle, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingolipids+inhibit+endosomal+recycling+of+nutrient+transporters+by+inactivating+ARF6.">Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6.</a> Journal of Cell Science. 131 (12), (2018).</li><li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APE1+upregulates+MMP-14+via+redox-sensitive+ARF6-mediated+recycling+to+promote+cell+invasion+of+esophageal+adenocarcinoma.">APE1 upregulates MMP-14 via redox-sensitive ARF6-mediated recycling to promote cell invasion of esophageal adenocarcinoma.</a> Cancer Research. 79 (17), 4426-4438 (2019).</li><li>Qi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arf6-driven+endocytic+recycling+of+CD147+determines+HCC+malignant+phenotypes.">Arf6-driven endocytic recycling of CD147 determines HCC malignant phenotypes.</a> Journal of Experimental and Clinical Cancer Research. 38 (1), 471 (2019).</li><li>Crupi, M. J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GGA3-mediated+recycling+of+the+RET+receptor+tyrosine+kinase+contributes+to+cell+migration+and+invasion.">GGA3-mediated recycling of the RET receptor tyrosine kinase contributes to cell migration and invasion.</a> Oncogene. 39 (6), 1361-1377 (2020).</li><li>Gamara, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Arf6+deletion+in+PLB-985+differentiated+in+neutrophil-like+cells+and+in+mouse+neutrophils:+impact+on+adhesion+and+migration.">Assessment of Arf6 deletion in PLB-985 differentiated in neutrophil-like cells and in mouse neutrophils: impact on adhesion and migration.</a> Mediators of Inflammation. 2020, 2713074 (2020).</li><li>Santy, L. C., Ravichandran, K. S., Casanova, J. 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+DOCK180/Elmo+complex+couples+ARNO-mediated+Arf6+activation+to+the+downstream+activation+of+Rac1.">The DOCK180/Elmo complex couples ARNO-mediated Arf6 activation to the downstream activation of Rac1.</a> Current Biology. 15 (19), 1749-1754 (2005).</li><li>Wibo, M., Dumont, J. E., Brown, B. L., Marshall, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fractionation+by+centrifugation+methods.">Cell fractionation by centrifugation methods.</a> Eukaryotic Cell Function and Growth: Regulation by Intracellular Cyclic Nucleotides. , 1-17 (1976).</li><li>Kelly, E. E., Horgan, C. P., McCaffrey, 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=Rab11+proteins+in+health+and+disease.">Rab11 proteins in health and disease.</a> Biochemical Society Transactions. 40 (6), 1360-1367 (2012).</li><li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+adaptor+FE65+stimulates+Rac1-mediated+neurite+outgrowth+by+recruiting+and+activating+ELMO1.">Neuronal adaptor FE65 stimulates Rac1-mediated neurite outgrowth by recruiting and activating ELMO1.</a> The Journal of Biological Chemistry. 293 (20), 7674-7688 (2018).</li><li>Chan, W. W. R., Li, W., Chang, R. C. C., Lau, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARF6-Rac1+signaling-mediated+neurite+outgrowth+is+potentiated+by+the+neuronal+adaptor+FE65+through+orchestrating+ARF6+and+ELMO1.">ARF6-Rac1 signaling-mediated neurite outgrowth is potentiated by the neuronal adaptor FE65 through orchestrating ARF6 and ELMO1.</a> FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology. 34 (12), 16397-16413 (2020).</li><li>Huber, L. A., Pfaller, K., Vietor, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+proteomics:+implications+for+subcellular+fractionation+in+proteomics.">Organelle proteomics: implications for subcellular fractionation in proteomics.</a> Circulation Research. 92 (9), 962-968 (2003).</li><li>Fleischer, S., Kervina, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+fractionation+of+rat+liver.">Subcellular fractionation of rat liver.</a> Methods in Enzymology. 31, 6-41 (1974).</li><li>Marsh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosome+and+lysosome+purification+by+free-flow+electrophoresis.">Endosome and lysosome purification by free-flow electrophoresis.</a> Methods in Cell Biology. 31, 319-334 (1989).</li><li>Stasyk, T., Huber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zooming+in:+fractionation+strategies+in+proteomics.">Zooming in: fractionation strategies in proteomics.</a> Proteomics. 4 (12), 3704-3716 (2004).</li><li>Iordachescu, A., Hulley, P., Grover, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+collection+of+nanoscopic+vesicles+from+an+organotypic+culture+model.">A novel method for the collection of nanoscopic vesicles from an organotypic culture model.</a> RSC Advances. 8 (14), 7622-7632 (2018).</li><li>Chavrier, P., vander Sluijs, P., Mishal, Z., Nagelkerken, B., Gorvel, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+endosome+membrane+dynamics+characterized+by+flow+cytometry.">Early endosome membrane dynamics characterized by flow cytometry.</a> Cytometry. 29 (1), 41-49 (1997).</li><li>Chasan, A. I., Beyer, M., Kurts, C., Burgdorf, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+specialized,+antigen-loaded+early+endosomal+subpopulation+by+flow+cytometry.">Isolation of a specialized, antigen-loaded early endosomal subpopulation by flow cytometry.</a> Methods in Molecular Biology. 960, 379-388 (2013).</li><li>Thapa, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-3-OH+kinase+signaling+is+spatially+organized+at+endosomal+compartments+by+microtubule-associated+protein+4.">Phosphatidylinositol-3-OH kinase signaling is spatially organized at endosomal compartments by microtubule-associated protein 4.</a> Nature Cell Biology. 22 (11), 1357-1370 (2020).</li><li>Guimaraes de Araujo, M. E., Fialka, I., Huber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Endocytic Organelles: Methods For Preparation And Analysis. In eLS. , (2001).</li><li>Rickwood, D., Graham, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Centrifugation Techniques. , (2015).</li><li>Lamberti, G., de Araujo, M. E., Huber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+macrophage+early+and+late+endosomes+by+latex+bead+internalization+and+density+gradient+centrifugation.">Isolation of macrophage early and late endosomes by latex bead internalization and density gradient centrifugation.</a> Cold Spring Harbor Protocols. 2015 (12), (2015).</li><li>Urbanska, A., Sadowski, L., Kalaidzidis, Y., Miaczynska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+APPL+endosomes:+the+role+of+annexin+A2+in+APPL+membrane+recruitment.">Biochemical characterization of APPL endosomes: the role of annexin A2 in APPL membrane recruitment.</a> Traffic. 12 (9), 1227-1241 (2011).</li></ol>

Ovenstående protokol skitserer procedurerne for isolering af genanvendelsesenomer fra dyrkede celler ved ultracentrifugering. Pålideligheden af denne metode er blevet påvist ved den seneste publikation22, der beviser, at genanvendelse endosomes er med succes isoleret fra andre organeller (figur 1), såsom Golgi apparatet og mitokondrier. Nogle kritiske skridt skal være opmærksomme på for at opnå et godt adskillelsesresultat. Under tilberedningen af saccharoseop...

Endosomal handel er en vigtig fysiologisk proces, der implicerer forskellige biologiske hændelser1, for eksempel transport af signalering receptorer, ion kanaler, og vedhæftning molekyler. Proteiner lokaliseret på plasmamembranen internaliseres ved endokytose2. De internaliserede proteiner sorteres derefter efter den tidlige endosome3. Nogle af proteinerne er målrettet mod lysosomer til nedbrydning4. Men en betydelig mængde proteiner genbruges tilbage til celleoverfladen ved hurtig genanvendelse og langsomme genanvendelsesprocesser. Ved hurtig genanvendelse forlader proteiner de tidlige endoomer og vender direkte tilbage til plasmamembranen. Omvendt sorteres proteiner først i langsom genanvendelse til det endoytiske genbrugsrum og transporteres derefter tilbage til plasmamembranen. Forskellige lastproteiner, for eksempel clathrin, retromer kompleks, retriever kompleks og Wiskott-Aldrich syndrom protein, og SCAR Homologue (WASH) kompleks, deltage i sådanne membran genanvendelse processer4,5,6,7,8,9. Balancen i endokytose og genanvendelse begivenhed er afgørende for celle overlevelse og bidrager til forskellige cellulære begivenheder10, for eksempel, celle vedhæftning, celle migration, celle polaritet, og signal transduktion.
ARF6, en lille GTPase, er en rapporteret regulator for endokritisk handel7,11,12. Af interesse har forskellige forskergrupper illustreret betydningen af ARF6 i endokritisk genanvendelse13,14,15,16,17. Undersøgelsen har til formål at undersøge forholdet mellem ARF6-medieret neurit udvækst og endokritisk genanvendelse. Den tidligere rapport tyder på, at aktiveringen af ARF6 er opstrøms til Rac1 aktivitet ved at handle på ELMO1-dedicator af cytokinesis 1 (DOCK180) kompleks18. Det er dog fortsat uklart, hvordan ARF6 udløser ELMO1-DOCK180-medieret Rac1-signalering. Density gradient ultracentrifugering blev anvendt til at undersøge den rolle, ARF6-medieret endokritisk genanvendelse i en sådan proces. Ved at bruge dette blev genanvendelses-endosome-holdige fraktion opnået fra cellelytter19. Fraktionen blev udsat for vestlig blotting for proteinindhold analyse. Immunoblot-resultaterne viste, at under tilstedeværelse af FE65, et hjerneberiget adapterprotein, øgede aktiv ARF6 betydeligt elmo1-niveauet i genanvendelses-endosomeholdig fraktion. Følgende protokol omfatter procedurerne for (1) transfecting pattedyrceller; 2) udarbejdelse af prøverne og densitetsgradientkolonnerne og (3) opnåelse af den genanvendelses-endosomeholdige fraktion.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL, Open-Top Thickwall Polypropylene Tube, 11 x 34 mm</td>
			<td>Beckman Coulter</td>
			<td>347287</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm tissue culture dish</td>
			<td>SPL</td>
			<td>20100</td>
			<td></td>
		</tr>
		<tr>
			<td>13.2 mL, Certified Free Open-Top Thinwall Ultra-Clear Tube, 14 x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>C14277</td>
			<td></td>
		</tr>
		<tr>
			<td>5x Sample Buffer</td>
			<td>GenScript</td>
			<td>MB01015</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>COX IV (3E11) Rabbit mAb</td>
			<td>Cell Signaling Technology</td>
			<td>4850S</td>
			<td>Rabbit monoclonal antibody for detecting COX IV.</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma-Aldrich</td>
			<td>C1988</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce Tissue Grinder, 7 mL</td>
			<td>DWK Life Sciences</td>
			<td>357542</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) with low glucose</td>
			<td>HyClone</td>
			<td>SH30021.01</td>
			<td></td>
		</tr>
		<tr>
			<td>ELMO1 antibody (B-7)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-271519</td>
			<td>Mouse monoclonal antibody for detecting ELMO1.</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>FE65 antibody (E-20)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-19751</td>
			<td>Goat polyclonal antibody for detecting FE65.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Research Grade</td>
			<td>HyClone</td>
			<td>SV30160.03</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH Monoclonal Antibody (6C5)</td>
			<td>Ambion</td>
			<td>AM4300</td>
			<td>Mouse monoclonal antibody for detecting GAPDH.</td>
		</tr>
		<tr>
			<td>ImageLab Software</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>Measurement of band intensity</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I2399</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal Anti-&#946;-COP antibody</td>
			<td>Sigma</td>
			<td>G6160</td>
			<td>Mouse monoclonal antibody for detecting &#946;-COP.</td>
		</tr>
		<tr>
			<td>Myc-tag (9B11) mouse mAb</td>
			<td>Cell Signaling Technology</td>
			<td>2276S</td>
			<td>Mouse monoclonal antibody for detecting myc tagged proteins.</td>
		</tr>
		<tr>
			<td>OmniPur EDTA, Disodium Salt, Dihydrate</td>
			<td>Calbiochem</td>
			<td>4010-OP</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima L-100 XP</td>
			<td>Beckman Coulter</td>
			<td>392050</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima MAX-TL</td>
			<td>Beckman Coulter</td>
			<td>A95761</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Media</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Tablets</td>
			<td>Gibco</td>
			<td>18912014</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosSTOP</td>
			<td>Roche</td>
			<td>4906845001</td>
			<td></td>
		</tr>
		<tr>
			<td>RAB11A-Specific Polyclonal antibody</td>
			<td>Proteintech</td>
			<td>20229-1-AP</td>
			<td>Rabbit polyclonal antibody for detecting Rab11.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Affymetrix</td>
			<td>AAJ21931A4</td>
			<td></td>
		</tr>
		<tr>
			<td>SW 41 Ti Swinging-Bucket Rotor</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
			<td></td>
		</tr>
		<tr>
			<td>TLA-120.2 Fixed-Angle Rotor</td>
			<td>Beckman Coulter</td>
			<td>362046</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Gibco</td>
			<td>25300062</td>
			<td></td>
		</tr>
	</tbody>
</table>

density gradient ultracentrifugation for investigating endocytic recycling in mammalian cells

Endosomal handel er en vigtig cellulær proces, der regulerer en bred vifte af biologiske begivenheder. Proteiner internaliseres fra plasmamembranen og transporteres derefter til de tidlige endoomer. De internaliserede proteiner kan overføres til lysosomet til nedbrydning eller genbruges tilbage til plasmamembranen. En robust endoytisk genbrugsvej er nødvendig for at afbalancere fjernelsen af membranmaterialer fra endokytose. Forskellige proteiner rapporteres at regulere vejen, herunder ADP-ribosylation faktor 6 (ARF6). Tæthedsgradient ultracentrifugering er en klassisk metode til cellefraktionering. Efter centrifugeringen er organeller sedimenteret på deres isopykiske overflade. Brøkene indsamles og bruges til andre downstream-applikationer. Beskrevet her er en protokol for at opnå en genanvendelse endosome-holdige fraktion fra transfected pattedyr celler ved hjælp af tæthed gradient ultracentrifugering. De isolerede fraktioner blev udsat for standard vestlige blotting for at analysere deres proteinindhold. Ved at anvende denne metode, identificerede vi, at plasmamembran målretning af opslugt og celle motilitet 1 (ELMO1), en Ras-relaterede C3 botulinum toksin substrat 1 (Rac1) guanine nukleotid udveksling faktor, er gennem ARF6-medieret endokritisk genanvendelse.

Efter at have fraktioneret de uoversendte HEK293-celler ved densitetsgradient ultracentrifugering blev der indsamlet 12 fraktioner startende fra toppen af gradienten. De høstede fraktioner blev fortyndet med fortyndingsbufferen i et 1:1-forhold og udsat for en anden centrifugeringsrunde. Prøverne blev derefter udsat for vestlige blotting for at analysere deres proteinindhold. Som vist i figur 1registreres genbrugsmarkøren Rab11 i fraktion 720. Andre subcellulære m...

Watch this Scientific Journal Video about Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells at JoVE.com

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

Dette papir har til formål at præsentere en protokol til forberedelse af genanvendelse endosomes fra pattedyr celler ved hjælp af saccharosetæthed gradient ultracentrifugering.

Tæthedsgradient ultracentrifugering til undersøgelse af endoytisk genanvendelse i pattedyrceller

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma ...

Denne protokol hjælper med at isolere genanvendelsesendosomer, der indeholder fraktion fra transfected pattedyrceller for at lette undersøgelsen af proteinhandel via endoytisk handel. Denne teknik er nem at håndtere og gælder for både vævs- og celleprøver. Demonstrere proceduren vil være Miss Zhai Yu Qi, en ph.d.-studerende fra Dr.Lao's Laboratory.Til at begynde med plade 2 gange 10 til de 6 celler i en 100 millimeter kultur skål. Den næste dag transfect cellerne med Lipofectamin i henhold til producentens anvisninger. For at høste cellerne skal du kassere kulturmediet 48 timer efter transfektion og vaske cellerne to gange med iskold PBS.Derefter tilsættes en milliliter iskold PBS suppleret med 0,5X protease hæmmer cocktail i 0,5X fosfatase hæmmer cocktail til hver skål. Dernæst ved hjælp af en celleskraber, indsamle cellerne og overføre celle suspension til en 15 milliliter centrifuge rør. cellerne ved centrifugering ved hjælp af en svingspandrotor.Kassér supernatanten og genbrug cellepillen forsigtigt i fem milliliter homogeniseringsbuffer. Saml cellerne igen ved centrifugering og resuspend celle pellet i en milliliter homogenisering buffer. Homogeniser cellerne med en Dounce Homogenizer med 15 til 20 slag.Overfør derefter homogonatet til et to milliliter centrifugeringsrør. Tilsæt 700 mikroliter homogeniseringsbuffer til homogonatet. Drej derefter den fortyndede homogonat og saml 1,5 milliliter af supernatanten.Drej den indsamlede supernatant igen, og saml derefter 1,4 milliliter af supernatanten og mærk den som post-nuklear supernatant. For at forberede tæthedsgradientkolonnen overføres 1,2 milliliter af det post-nukleare supernatant til et ultracentrifugerør. Tilsæt derefter en milliliter 62% saccharoseopløsning til prøven og bland godt ved skånsom pipettering.Derefter tilsættes forsigtigt 3,3 milliliter 35% saccharoseopløsning oven på prøven, efterfulgt af 2,2 milliliter 25% saccharoseopløsning oven på 35% saccharoseopløsningen. Endelig fyldes ultracentrifugerøret til toppen med homogeniseringsbuffer. Centrifugere tætheden gradient kolonne og omhyggeligt indsamle 12 fraktioner af 1 milliliter hver, startende fra toppen af gradient.Derefter fortyndes alle fraktioner med en milliliter fortyndingsbuffer og centrifugerer de fortyndede prøver. Efter centrifugering, aspirere supernatant og tilsæt 50 mikroliter af 1X prøve buffer til at høste fraktioner. Genbrugsmarkøren Rab11 blev fundet i fraktion syv.Andre subcellulære markører, herunder beta COP, COX IV, GAPDH, EEA1, Rab 7 og Lamp1 blev også undersøgt. Der blev også fundet et positivt EØS1-signal i fraktion syv. De målte båndintensiteter gapdh, Cox IV og Rab11 i både fraktion syv og post-nukleare supernatant er vist her.Efter at have transecting HEC293 celler med enten ELMO1, ELMO1 ARF6Q67L, eller ELMO1 ARF6Q67L FE65, ingen ændringer blev fundet i ELMO1 distribution med ARF6Q67L og FE65 overekspression. ELMO1-niveauet i fraktion syv blev sammenlignet mellem forskellige transinfektioner og viste sig at være forhøjet efter cotransfection af ARF6Q67L. Der blev observeret en yderligere stigning, da både ARF6Q67L og FE65 blev samtil udtrykt.Omvendt, banke ud FE65 mindsket ARF6 medieret Elmo1 berigelse i fraktion syv. Den opnåede brøk kan anvendes til efterfølgende analyse. For eksempel, vestlige blotting at visualisere protein niveau ændringer i fraktionen.

Research

JoVE Journal

Biochemistry

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The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

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This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

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