This work was supported by a grant from the Canadian Institutes for Health Research (MOP-86514).

Les procédures expérimentales sur des souris ont été approuvés par le Comité de l&#39;Université McGill (protocole 4966) de protection des animaux. 1. Préparation d&#39;extraits de protéine à partir de cellules cultivées <ol><li> Laver les cellules en culture à deux reprises avec 10 ml de phosphate refroidie à la glace une solution saline tamponnée (PBS). </li><li> Grattez cellules adhérentes soit avec une spatule en caoutchouc ou d&#39;un grattoir à cellules en plastique dans 1 ml de PBS, le transfert suspension glacée dans un tube de 1,5 ml. </li><li> Centrifugeuse à 5 min à 700 xg, à 4 ° C. Aspirer PBS. </li><li> Ajouter 100 pi de tampon de lyse cytoplasmique glacée (Tableau 1) par 10 7 cellules, et la pipette de haut en bas. </li><li> Incuber sur de la glace pendant 20 min. </li><li> Spin pendant 10 min à pleine vitesse dans une microcentrifugeuse à 4 ° C. </li><li> Jeter culot. Transférer le surnageant dans un nouveau tube de 1,5 ml et garder sur la glace. </li><li> Determine concentration de protéine (généralement de 1 à 10 ug / ul) en utilisant le 23 dosage de Bradford. </li><li> Aliquoter et cellulaires magasin extraits à -80 ° C jusqu&#39;à utilisation. </li></ol> 2. Préparation d&#39;extraits de protéine à partir de foie et la rate de souris <ol><li> Euthanasier une souris avec inhalation de CO 2. </li><li> Placez l&#39;animal euthanasié sur un tapis propre sur une planche de dissection. Ouvrez l&#39;abdomen avec des ciseaux. </li><li> Disséquer le foie et la rate à l&#39;aide des ciseaux et des pinces, et rincer chaque tissu dans du PBS environ 50 ml de glace-froid. </li><li> Tissus en petits morceaux immédiatement couper avec un scalpel (par exemple: environ 1-2 mm 3). </li><li> Sans délai, mettre des morceaux de tissus dans une nouvelle cryotube puis enclenchez les congeler dans l&#39;azote liquide. Magasin enfichable aliquotes congelées de tissus à -80 ° C jusqu&#39;à utilisation. </li><li> Homogénéiser une seule pièce de tissu congelé (environ 1-2 mm 3) 0,25 au 0,5 ml deglacé tampon de lyse cytoplasmique (tableau 1) avec un homogénéisateur de tissu pendant 10 sec. </li><li> Transfert homogénat à 1,5 ml microtube et le froid sur la glace pendant 20 min. </li><li> Spin pendant 10 min à pleine vitesse dans une microcentrifugeuse à 4 ° C. </li><li> Jeter culot et le transfert surnageant dans un nouveau tube de 1,5 ml. Garder sur la glace. </li><li> Déterminer la concentration en protéine (généralement de 1 à 10 ug / ul) en utilisant le 23 dosage de Bradford. </li><li> Aliquoter et cellulaires magasin extraits à -80 ° C jusqu&#39;à utilisation. </li></ol> 3. Préparation de Radiolabeled IRE-sonde <ol><li> Linéariser le plasmide contenant IRE I-12.CAT 22 par incubation à 37 ° C pendant 1 heure avec l&#39;endonucléase de restriction XbaI (1 U par pg de plasmide), qui clive en aval de la séquence IRE. Le plasmide linéarisé est utilisé comme matrice pour la transcription in vitro. </li><li> Mettre en place unn in vitro réaction de transcription dans un volume total de 20 ul. Utiliser les solutions mères indiqués dans le tableau 2 et ajouter: 1 ul de modèles de plasmide linéarisé, un tampon de transcription 4 pi; 1 pl de mélange ATP / CTP / GTP mélange, 10 ul de [α- 32 P] -UTP, 2 pl de dithiothréitol, un inhibiteur de RNase et 1 pi 1 pi d&#39;ARN polymerase de T7. Mélanger en pipetant. </li><li> Incuber à 40 ° C pendant 1 h 24. </li></ol> 4. Purification de Radiolabeled IRE-sonde <ol><li> Mettre fin à la réaction de transcription in vitro par addition de 1 pi d&#39;EDTA 0,5 M, pH 8. Mélanger en pipetant de haut en bas. </li><li> Ajouter 10 ul de 10 mg / ml d&#39;ARNt, en tant que support pour une meilleure précipitation. Mélanger en pipetant. </li><li> Ajouter 82,5 ul de 3 M d&#39;acétate d&#39;ammonium. Mélanger au vortex. </li><li> Ajouter 273 ul d&#39;éthanol. Mélanger au vortex. </li><li> Laisser reposer à température ambiante pendant 5 min. </li><li> Spin pendant 10 min à pleine vitesse dans une microcentrifugeuse à température ambiante. Surnageant défausse. </li><li> Laver culot avec 100 pi d&#39;éthanol à 70%. </li><li> Spin pendant 10 min à pleine vitesse dans une microcentrifugeuse à température ambiante. Surnageant défausse. </li><li> Air culot sec pendant 10 min. </li><li> Resuspendre le culot dans 100 pi de double distillée, préalablement autoclave H 2 O. </li><li> Quantifier la radioactivité dans un compteur à scintillation liquide, sonde IRE aliquote de radiomarqué et conserver à -80 ° C jusqu&#39;à utilisation. Aliquots congelés peuvent être utilisés pour 3 semaines. </li></ol> 5. Préparation d&#39;un gel de Polyacrylamide natif à EMSA <ol><li> Assemblez le gel (16 x 16 cm) en utilisant 1,5 entretoises mm et peigne. </li><li> Pour préparer un gel de Polyacrylamide natif à 6%, utiliser les solutions mères indiqués dans le tableau 3 Mélanger 7,5 ml de 40% d&#39;acrylamide. Bisacrylamide, 5 ml de TBE 5x et 37,5 mlDouble H 2 O. distillée </li><li> Ajouter 0,5 ml de 10% persulfate fraîchement préparée d&#39;ammonium (APS) et 25 ul tétraméthyléthylènediamine (TEMED). </li><li> Verser immédiatement la solution d&#39;acrylamide au gel et laissez polymériser. Attendez environ 30 min. </li><li> Monter l&#39;appareil d&#39;électrophorèse avec le gel, remplir les réservoirs avec 0,5x TBE et se connecter avec l&#39;alimentation. </li></ol> 6. mobilité électrophorétique Maj Assay <ol><li> Diluer 25 pg d&#39;extrait de protéine à partir de cellules ou de tissus avec cytoplasmique tampon de lyse (tableau 1) dans un volume total de 10 ul en (concentrations de protéine plus faibles peuvent également être utilisés). Garder sur la glace. Si nécessaire, ajouter 1 pl de 1: 4 dilué 2-mercaptoéthanol (2-ME) pour activer IRP1 dormance (concentration finale: 2%) 25. </li><li> Diluer la sonde IRE radiomarqué en chambre double H 2 O distillée à 200 000 cpm / ul, dénaturation thermique à 95 °C pendant 1 min, et refroidir à la température ambiante pendant au moins 5 min. </li><li> Mettre en place une réaction EMSA en ajoutant 1 pl de la sonde IRE radiomarqué à l&#39;extrait de protéine. </li><li> Incuber pendant 20 min à température ambiante. </li><li> Ajouter 1 pi de 50 mg / ml d&#39;héparine à la réaction (à inhiber les interactions protéiques non spécifiques avec la sonde 9) et poursuivre l&#39;incubation pendant encore 10 min. Aliquoter la solution mère de l&#39;héparine (50 mg / ml) et conserver à -80 ° C. </li><li> Lors de l&#39;utilisation de longues sondes radiomarquées (&gt; 60 nucleotides), ajouter 1 pl de RNase T1 (1 U / ul) et incuber pendant 10 min à température ambiante pour réduire la protéine non spécifique à la liaison à la sonde, et pour permettre une meilleure séparation de l&#39;ARN / protéine complexe au cours de l&#39;électrophorèse. </li><li> Ajouter 3 pi de tampon de chargement (80% de glycérol + bleu bromophénol), mélanger et la charge sur le gel de polyacrylamide non dénaturant 6%. </li><li> Exécutez le gel pendant 60 min à 130 V (5 V / cm). </li><li> Transfert du gel sur un grand papier filtre et sèche. </li><li> Exposer à un film et développer autoradiographie. Temps d&#39;exposition peut varier de 1 h (ou moins) jusqu&#39;à O / N. </li></ol>

<ol>
	<li>Fried, M., Crothers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equilibria+and+kinetics+of+lac+repressor-operator+interactions+by+polyacrylamide+gel+electrophoresis.">Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis.</a> Nucleic Acids Res. 9, 6505-6525 (1981).</li><li>Garner, M. M., Revzin, 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+gel+electrophoresis+method+for+quantifying+the+binding+of+proteins+to+specific+DNA+regions:+application+to+components+of+the+Escherichia+coli+lactose+operon+regulatory+system.">A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system.</a> Nucleic Acids Res. 9, 3047-3060 (1981).</li><li>Ryder, S. P., Recht, M. I., Williamson, J. R. <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+protein-RNA+interactions+by+gel+mobility+shift.">Quantitative analysis of protein-RNA interactions by gel mobility shift.</a> Methods Mol Biol. 488, 99-115 (2008).</li><li>Hellman, L. M., Fried, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+mobility+shift+assay+(EMSA)+for+detecting+protein-nucleic+acid+interactions.">Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.</a> Nat Protoc. 2, 1849-1861 (2007).</li><li>Luscieti, 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=Novel+mutations+in+the+ferritin-L+iron-responsive+element+that+only+mildly+impair+IRP+binding+cause+hereditary+hyperferritinaemia+cataract+syndrome.">Novel mutations in the ferritin-L iron-responsive element that only mildly impair IRP binding cause hereditary hyperferritinaemia cataract syndrome.</a> Orphanet J Rare Dis. 8, 30 (2013).</li><li>Rio, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Filter-binding assay for analysis of RNA-protein interactions. 2012, 1078-1081 (2012).</li><li>Wang, 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=Iron-mediated+degradation+of+IRP2:+an+unexpected+pathway+involving+a+2-oxoglutarate-dependent+oxygenase+activity.">Iron-mediated degradation of IRP2: an unexpected pathway involving a 2-oxoglutarate-dependent oxygenase activity.</a> Mol. Cell. Biol. 24, 954-965 (2004).</li><li>Leibold, E. A., Munro, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+protein+binds+in+vitro+to+a+highly+conserved+sequence+in+the+5'+untranslated+regions+of+ferritin+heavy-+and+light-subunit+mRNAs.">Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5' untranslated regions of ferritin heavy- and light-subunit mRNAs.</a> Proc. Natl. Acad. Sci. USA. 85, 2171-2175 (1988).</li><li>Haile, D. J., Hentze, M. W., Rouault, T. A., Harford, J. B., Klausner, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+interaction+of+the+iron-responsive+element+binding+protein+with+iron-responsive+RNA+elements.">Regulation of interaction of the iron-responsive element binding protein with iron-responsive RNA elements.</a> Mol. Cell. Biol. 9, 5055-5061 (1989).</li><li>Mueller, S., Pantopoulos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+iron+regulatory+protein-1+(IRP1)+by+oxidative+stress.">Activation of iron regulatory protein-1 (IRP1) by oxidative stress.</a> Methods Enzymol. 348, 324-337 (2002).</li><li>Wang, J., Pantopoulos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cellular+iron+metabolism.">Regulation of cellular iron metabolism.</a> Biochem J. 434, 365-381 (2011).</li><li>Hentze, M. 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=Identification+of+the+iron-responsive+element+for+the+translational+regulation+of+human+ferritin+mRNA.">Identification of the iron-responsive element for the translational regulation of human ferritin mRNA.</a> Science. 238, 1570-1573 (1987).</li><li>Casey, J. L., 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=Iron-responsive+elements:+regulatory+RNA+sequences+that+control+mRNA+levels+and+translation.">Iron-responsive elements: regulatory RNA sequences that control mRNA levels and translation.</a> Science. 240, 924-928 (1988).</li><li>Dandekar, 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=Identification+of+a+novel+iron-responsive+element+in+murine+and+human+erythroid+d-aminolevulinic+acid+synthase+mRNA.">Identification of a novel iron-responsive element in murine and human erythroid d-aminolevulinic acid synthase mRNA.</a> EMBO J. 10, 1903-1909 (1991).</li><li>Gray, N. K., Pantopoulos, K., Dandekar, T., Ackrell, B. A. C., Hentze, 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=Translational+regulation+of+mammalian+and+drosophila+citric+acid+cycle+enzymes+via+iron-responsive+elements.">Translational regulation of mammalian and drosophila citric acid cycle enzymes via iron-responsive elements.</a> Proc. Natl. Acad. Sci. USA. 93, 4925-4930 (1996).</li><li>McKie, A. 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=A+novel+duodenal+iron-regulated+transporter+IREG1,+implicated+in+the+basolateral+transfer+of+iron+to+the+circulation.">A novel duodenal iron-regulated transporter IREG1, implicated in the basolateral transfer of iron to the circulation.</a> Mol. Cell. 5, 299-309 (2000).</li><li>Gunshin, 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=Cloning+and+characterization+of+a+mammalian+protein-coupled+metal-ion+transporter.">Cloning and characterization of a mammalian protein-coupled metal-ion transporter.</a> Nature. 388, 482-488 (1997).</li><li>Sanchez, M., Galy, B., Muckenthaler, M. U., Hentze, 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=Iron-regulatory+proteins+limit+hypoxia-inducible+factor-2alpha+expression+in+iron+deficiency.">Iron-regulatory proteins limit hypoxia-inducible factor-2alpha expression in iron deficiency.</a> Nat. Struct. Mol. Biol. 14, 420-426 (2007).</li><li>Sanchez, M., 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=Iron+regulation+and+the+cell+cycle:+Identification+of+an+iron-responsive+element+in+the+3'-untranslated+region+of+human+cell+division+cycle+14A+mRNA+by+a+refined+microarray-based+screening+strategy.">Iron regulation and the cell cycle: Identification of an iron-responsive element in the 3'-untranslated region of human cell division cycle 14A mRNA by a refined microarray-based screening strategy.</a> J. Biol. Chem. 281, 22865-22874 (2006).</li><li>Santos, C. O., 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=An+iron+responsive+element-like+stem-loop+regulates+alpha-hemoglobin-stabilizing+protein+mRNA.">An iron responsive element-like stem-loop regulates alpha-hemoglobin-stabilizing protein mRNA.</a> J Biol Chem. 283, 26956-26964 (2008).</li><li>Liu, Z., 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=Siderophore-mediated+iron+trafficking+in+humans+is+regulated+by+iron.">Siderophore-mediated iron trafficking in humans is regulated by iron.</a> J Mol Med (Berl. 90, 1209-1221 (2012).</li><li>Gray, N. K., 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=Recombinant+iron+regulatory+factor+functions+as+an+iron-responsive+element-binding+protein,+a+translational+repressor+and+an+aconitase.+A+functional+assay+for+translational+repression+and+direct+demonstration+of+the+iron+switch.">Recombinant iron regulatory factor functions as an iron-responsive element-binding protein, a translational repressor and an aconitase. A functional assay for translational repression and direct demonstration of the iron switch.</a> Eur. J. Biochem. 218, 657-667 (1993).</li><li>Bradford, M. 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+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Testa, U., 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=Differential+regulation+of+iron+regulatory+element-binding+protein(s)+in+cell+extracts+of+activated+lymphocytes+versus+monocytes-macrophages.">Differential regulation of iron regulatory element-binding protein(s) in cell extracts of activated lymphocytes versus monocytes-macrophages.</a> J. Biol. Chem. 266, 13925-13930 (1991).</li><li>Hentze, M. W., Rouault, T. A., Harford, J. B., Klausner, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation-reduction+and+the+molecular+mechanism+of+a+regulated+RNA-protein+interaction.">Oxidation-reduction and the molecular mechanism of a regulated RNA-protein interaction.</a> Science. 244, 357-359 (1989).</li><li>Meyron-Holtz, E. G., 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=Genetic+ablations+of+iron+regulatory+proteins+1+and+2+reveal+why+iron+regulatory+protein+2+dominates+iron+homeostasis.">Genetic ablations of iron regulatory proteins 1 and 2 reveal why iron regulatory protein 2 dominates iron homeostasis.</a> EMBO J. 23, 386-395 (2004).</li><li>Huang, F. W., Pinkus, J. L., Pinkus, G. S., Fleming, M. D., Andrews, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+juvenile+hemochromatosis.">A mouse model of juvenile hemochromatosis.</a> J. Clin. Invest. 115, 2187-2191 (2005).</li><li>Sebastiani, G., Pantopoulos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorders+associated+with+systemic+or+local+iron+overload:+from+pathophysiology+to+clinical+practice.">Disorders associated with systemic or local iron overload: from pathophysiology to clinical practice.</a> Metallomics. 3, 971-986 (2011).</li><li>Galy, B., Ferring, D., Hentze, 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=Generation+of+conditional+alleles+of+the+murine+iron+regulatory+protein+(IRP)-1+and+-2+genes.">Generation of conditional alleles of the murine iron regulatory protein (IRP)-1 and -2 genes.</a> Genesis. 43, 181-188 (2005).</li><li>Gkouvatsos, K., 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=Iron-dependent+regulation+of+hepcidin+in+Hjv-/-+mice:+Evidence+that+hemojuvelin+is+dispensable+for+sensing+body+iron+levels.">Iron-dependent regulation of hepcidin in Hjv-/- mice: Evidence that hemojuvelin is dispensable for sensing body iron levels.</a> PLoS ONE. 9, 85530 (2014).</li><li>Goforth, J. B., Anderson, S. A., Nizzi, C. P., Eisenstein, 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=Multiple+determinants+within+iron-responsive+elements+dictate+iron+regulatory+protein+binding+and+regulatory+hierarchy.">Multiple determinants within iron-responsive elements dictate iron regulatory protein binding and regulatory hierarchy.</a> RNA. 16, 154-169 (2010).</li><li>Gopinath, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+RNA-protein+interactions.">Mapping of RNA-protein interactions.</a> Anal Chim Acta. 636, 117-128 (2009).</li></ol>

The authors declare that they have no competing financial interests.

Ici, nous décrivons un protocole qui a été mis au point pour étudier les activités IRE contraignants de IRP1 et IRP2, et nous montrons des données représentatives. En utilisant différentes sondes, ce protocole peut également être ajustée pour l&#39;étude d&#39;autres protéines liant l&#39;ARN. Une étape critique est la taille de la sonde. L&#39;utilisation de sondes longues, ce qui est fréquent lorsque le site de liaison exacte est inconnue, peut entraîner des complexes ARN / protéine qui ne migrent pas...

L&#39;EMSA a été initialement développé pour étudier l&#39;association de protéines de liaison à l&#39;ADN avec des séquences d&#39;ADN cible 1,2. Le principe est similaire pour les interactions ARN / protéine 3, qui est l&#39;objet de cet article. En bref, l&#39;ARN est chargé négativement et migrer vers l&#39;anode pendant l&#39;électrophorèse non dénaturant dans polyacrylamide (ou gels agarose). La migration dans le gel dépend de la taille de l&#39;ARN, qui est proportionnelle à la charge. La liaison d&#39;une protéine spécifique à l&#39;ARN modifie sa mobilité, et les complexes migre plus lentement par rapport à l&#39;ARN libre. Ceci est principalement dû à une augmentation de la masse moléculaire, mais aussi à des altérations de la charge et éventuellement conformation. Utilisant un ARN marqué comme sonde permet un contrôle facile de la «retard de gel&quot; ou &quot;bandshift&quot;. L&#39;utilisation de 32 sondes d&#39;ARN P marqué est très répandue et offre une sensibilité élevée. La migration des ARN / complexes de protéines et d&#39;ARN libre sont détectéspar autoradiographie. Inconvénients sont la demi-vie courte de 32 P (14,29 jours), la détérioration progressive de la qualité de la sonde due à la radiolyse, l&#39;exigence d&#39;une licence de la radioactivité et de l&#39;infrastructure pour les travaux de la radioactivité, et de biosécurité préoccupations potentielles. Par conséquent, les méthodes non isotopiques alternatives pour marquage de la sonde d&#39;ARN ont été développés, par exemple avec de la biotine ou des fluorophores, qui permettent une détection par imagerie de fluorescence ou de chimioluminescence 4,5. Les limitations de ces procédés sont le coût plus élevé et souvent une sensibilité réduite par rapport à marquage isotopique et le potentiel de marqueurs non isotopiques pour interférer avec l&#39;interaction ARN / protéine. Les non-dénaturantes des gels de Polyacrylamide sont appropriés pour la plupart des applications EMSA et sont couramment utilisés. À l&#39;occasion, des gels d&#39;agarose peuvent représenter une alternative pour l&#39;analyse de grands complexes. Le principal avantage de l&#39;EMSA est qu&#39;il combine la simplicité, la sensibilité et la robustesse 4 &lt;/ Sup&gt;. Le test peut être réalisé en quelques heures et ne nécessite pas une instrumentation sophistiquée. Les interactions ARN / protéine peut être détectée par l&#39;EMSA à des concentrations aussi faibles que 0,1 nm ou moins, et dans une large gamme de conditions de liaison (pH 4,0 à 9,5, monovalent concentration en sel de 1 à 300 mM, et la température de 0 à 60 ° C). ARN / protéines formation du complexe peut également être étudié par le test de filtre contraignant. Ce est une procédure simple, rapide et peu coûteux basé sur la conservation de complexes ARN / protéines dans un filtre de nitrocellulose, tandis qu&#39;une sonde d&#39;ARN libre passe par six. Par rapport à l&#39;EMSA, elle est limitée dans les cas où la sonde d&#39;ARN contient de multiples sites de liaison, ou l&#39;extrait brut contient plus d&#39;une des protéines liant l&#39;ARN qui se lient à la sonde sur le même site. Alors que de multiples interactions ARN / protéines seront échapper à la détection par le test de filtre contraignantes, elles peuvent être facilement visualisés par l&#39;EMSA. Dans certains cas, la visualisation est veillen possible lorsque deux complexes ARN / protéines co-émigrer (par exemple, IRP1 humaine / IRE et IRP2 / complexes IRE), en ajoutant un anticorps dirigé contre l&#39;une des protéines liant l&#39;ARN à la réaction EMSA, ce qui donne encore un retard sur le gel ( &quot;supershift&quot;) 7. L&#39;EMSA a été largement utilisé pour étudier IRP1 et IRP2, qui sont des régulateurs post-transcriptionnel du métabolisme du fer 8-10. Ils fonctionnent en se liant à IRE, structures en épingle à cheveux phylogénétiquement conservés dans les UTR de plusieurs ARNm 11. IRE ont été découverts dans les ARNm codant la ferritine 12 et récepteur de la transferrine 1 (TfR1) 13, les protéines de stockage du fer et de l&#39;absorption, respectivement. Plus tard, IRE ont été trouvés dans érythroïde spécifique aminolévulinate synthase (ALAS2) 14, aconitase mitochondriale 15, 16 ferroportine, divalent transporteur de métaux 1 (DMT1) 17, l&#39;hypoxie inducible factor 2 <em&gt; α (HIF2α) 18, et d&#39;autres ARNm 19-21. Le H- prototype et ARNm L-ferritine contiennent une IRE dans leur 5 &#39;UTR, tandis que TfR1 ARNm contient plusieurs IRE à son extrémité 3&#39; UTR. Interactions IRE / IRP spécifiquement inhiber la traduction de l&#39;ARNm de la ferritine en bloquant stériquement son association des sous-unités 43S ribosomique; en outre, ils stabilisent TfR1 ARNm contre clivage endonucléolytique. IRP1 et la part de IRP2 vaste similarité de séquence et de présenter une activité élevée de IRE-contraignant dans les cellules de fer-affamés. Dans les cellules de fer-remplie, IRP1 assemble un cluster Fe-S cubane qu&#39;il convertit en aconitase cytosolique au détriment de son activité IRE contraignant, tandis que IRP2 subit une dégradation par le protéasome. Ainsi, les interactions IRE / IRP dépendent du statut en fer cellulaire, mais sont également régies par d&#39;autres signaux, tels que H 2 O 2, l&#39;oxyde nitrique (NO) ou hypoxie. Ici, nous décrivons le protocole pour évaluer l&#39;activité IRE liaison à partir d&#39;extraits cellulaires bruts et de tissus par EMSA. Nous avons utilisé une sonde IRE marqué au 32P H-ferritine qui a été généré par transcription in vitro à partir d&#39;une matrice d&#39;ADN plasmidique (de I-12.CAT), où la séquence IRE a été initialement introduit dans l&#39;orientation sens en aval du site de l&#39;ARN polymerase T7 par clonage des oligonucleotides synthétiques annelés 22.

<table border="0" cellpadding="0" cellspacing="0" width="430">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:195px;">Name of the Materials&#160;</td>
			<td style="width:119px;">Company</td>
			<td style="width:117px;">Catalog Number&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">leupeptin</td>
			<td>SIGMA</td>
			<td>L2884</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PMSF</td>
			<td>SIGMA</td>
			<td align="right">78830</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BioRad Protein Assay</td>
			<td>BIORAD</td>
			<td>500-0006</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">T7 RNA polymerase</td>
			<td>Thermoscientific</td>
			<td>EPO111</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rnase Inhibitor</td>
			<td>Invitrogen</td>
			<td>15518-012</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">UTP [alpha-32P]</td>
			<td>Perkin-Elmer</td>
			<td>NEG507H</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scintillation liquid</td>
			<td>Beckman Coulter</td>
			<td align="right">141349</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">heparin</td>
			<td>SIGMA</td>
			<td>H0777</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rnase T1</td>
			<td>Thermoscientific</td>
			<td>EN0541</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Name of the Equipments&#160;</td>
			<td>Company</td>
			<td>Catalog Number&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue Ruptor</td>
			<td>Qiagen</td>
			<td align="right">9001271</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scintillation counter</td>
			<td>Beckman Coulter</td>
			<td>LS6500</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Protean II xi Cell&#160;&#160;</td>
			<td>BIORAD</td>
			<td>165-1834</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">20 wells combs 1.5mm thick</td>
			<td>BIORAD</td>
			<td>165-1868</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1.5 mm spacers</td>
			<td>BIORAD</td>
			<td>165-1849</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PowerPac</td>
			<td>BIORAD</td>
			<td>164-5070</td>
		</tr>
	</tbody>
</table>

electrophoretic mobility shift assay (emsa) for the study of rna-protein interactions: the ire/irp example

Interactions ARN / protéines sont essentielles pour voies de régulation post-transcriptionnel. Parmi les protéines liant l&#39;ARN cytosoliques les mieux caractérisés sont des protéines régulatrices du fer, et IRP1 IRP2. Ils se lient à repasser éléments sensibles (IRES) dans les régions non traduites (UTR) de plusieurs ARNm cibles, contrôlant ainsi la traduction ou la stabilité des ARNm. Interactions IRE / IRP ont été largement étudiés par l&#39;EMSA. Ici, nous décrivons le protocole EMSA pour analyser l&#39;activité IRE contraignant de IRP1 et IRP2, qui peut être généralisée à évaluer l&#39;activité d&#39;autres protéines liant l&#39;ARN ainsi. Un lysat de protéine brute contenant une protéine de liaison à l&#39;ARN, ou une préparation purifiée de cette protéine, est incubé avec un excès de 32 sonde d&#39;ARN marquée au P, ce qui permet la formation du complexe. L&#39;héparine est ajoutée à empêcher protéine non spécifique de liaison à la sonde. Ensuite, le mélange est analysé par électrophorèse non dénaturante sur un gel de Polyacrylamide. La sonde libremigre rapide, tandis que l&#39;ARN / protéines expositions complexes de mobilité retardée; par conséquent, la procédure est aussi appelé «retard de gel&quot; ou le dosage &quot;de bandshift&quot;. Après achèvement de l&#39;électrophorèse, le gel est séché et complexes ARN / protéine, ainsi que la sonde libre, sont détectés par autoradiographie. L&#39;objectif global du protocole est de détecter et de quantifier IRE / IRP et d&#39;autres interactions ARN / protéines. En outre, l&#39;EMSA peut également être utilisé pour déterminer la spécificité, l&#39;affinité de liaison, et la stoechiométrie de l&#39;interaction ARN / protéine à l&#39;étude.

Une sonde IRE radiomarqué a été préparé, comme décrit dans les sections 3 et 4 du protocole. La séquence de la sonde était 5&#39;-GGGCGAAUUC GAGCUCGGUA CCCGGGGAUC CUG C UUCAA C AGUGC UUGGA CGGAUCCU-3 &#39;; les nucléotides gras représentent un résidu C non apparié et la boucle, qui sont caractéristiques IRE critiques. La radioactivité spécifique de la sonde est de 4,5 x 10 9 cpm / ug d&#39;ARN. Pour évaluer les effets des perturbat...

Watch this Scientific Journal Video about Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example at JoVE.com

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Nous présentons ici un protocole d&#39;analyser les interactions ARN / protéines. L&#39;essai de décalage de mobilité électrophorétique (EMSA) est basé sur la migration différentielle des complexes ARN / ARN et de protéine libre pendant l&#39;électrophorèse sur gel natif. En utilisant une sonde d&#39;ARN radiomarqué, complexes / protéine ARN peuvent être visualisés par autoradiographie.

Maj de mobilité électrophorétique Assay (EMSA) pour l&#39;étude des interactions ARN-protéine: L&#39;IRE / IRP Exemple

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

electrophoretic-mobility-shift-assay-emsa-for-study-rna-protein

Research

JoVE Journal

Biology

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

53.2K vues.  Jewish General Hospital.  Nous présentons ici un protocole d'analyser les interactions ARN / protéines. L'essai de décalage de mobilité électrophorétique (EMSA) est basé sur la migration différentielle des complexes ARN / ARN et de protéine libre pendant l'électrophorèse sur gel natif. En utilisant une sonde d'ARN radiomarqué, complexes / protéine ARN peuvent être visualisés par autoradiographie. 

Vidéo: Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test sensibilité visuelle à la vitesse et la direction du mouvement chez les lézards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Recherche

Biologie

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Optimisé fibrine billes de gel d&#39;analyse pour l&#39;étude de l&#39;angiogenèse

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Computer-Generated Stimuli modèle animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself dispositif de récupération d&#39;échantillons cryoconservés tombé accidentellement dans les réservoirs de stockage cryogéniques

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Un essai en phase liquide d&#39;affinité de capture utilisant des billes magnétiques pour étudier interaction protéine-protéine: Le poliovirus Exemple-Nanobodies

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

La protéine plate-forme de production complexe MultiBac à l&#39;EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Bourgeonnement de levure extraction et purification des protéines pour l&#39;étude de la fonction, Interactions, et des modifications post-traductionnelles

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

La combinaison unique molécule manipulation et d&#39;imagerie pour l&#39;étude des interactions protéine-ADN

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Immunoprécipitation de la chromatine test pour l&#39;identification de Arabidopsis interactions protéine-ADN<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Le dépistage de la fonctionnelle non-codant des variants génétiques utilisant mobilité électrophorétique Maj Assay (EMSA) et l&#39;ADN-affinité Précipitations Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...