This work was supported by NIH grants (NS070577 and NS084869).

Remarque: La composition de toutes les solutions citées ci-dessous peut être trouvé dans le tableau Matériaux / Équipement. 1. Protéine A Colonne Antibody Purification <ol><li> Equilibrer une colonne de protéine de spin et de tous les tampons nécessaires à la température ambiante pendant 15 minutes avant de commencer la procédure. </li><li> Diluer le sérum immun 1: 1 avec de l&#39;IgG de la protéine A Binding Buffer. (5 ml de sérum est recommandé). </li><li> Tapoter doucement une colonne de protéine de spin sur la paillasse pour déloger toute résine qui peut être logé dans le capuchon. Retirez le couvercle supérieur et enclenchez doucement la fermeture de fond. Placer la colonne dans un tube collecteur de 15 ml et laisser la solution de stockage se vider. </li><li> Ajouter 5 ml de protéine A IgG Binding Buffer et laissez la solution se vider. </li><li> Appliquer le sérum immun dilué dans la colonne et recueillir l&#39;écoulement. Pour de meilleurs résultats, ajouter un volume d&#39;échantillon qui contient une concentration d&#39;anticorps inférieure à 80% d&#39;anticorps se liant à la capacit de la colonney. </li><li> Laver la colonne avec 15 ml de protéine A ou IgG Binding Buffer jusqu&#39;à ce que l&#39;absorbance à 280 nm est inférieur à 0,1. </li><li> Ajouter 100 pi de tampon de neutralisation à cinq tubes de prélèvement étiquetés. </li><li> Ajouter 5 ml de tampon d&#39;élution d&#39;IgG de la protéine A et la colonne recueillir fractions de 1 ml dans chacun des tubes contenant 100 pi de tampon de neutralisation. </li><li> Mesurer l&#39;absorption de chaque fraction à 280 nm et des fractions piscine avec une absorbance supérieure à 0,5. Gardez les fractions regroupées sur la glace jusqu&#39;à ce que prêt pour la dialyse. </li><li> Régénérer la colonne en ajoutant 8 ml d&#39;IgG tampon d&#39;élution et laissez la solution de circuler à travers la colonne. </li><li> Rincer la colonne avec une solution Binding Protein A jusqu&#39;à ce que les IgG éluant pH revient à 7.4. </li><li> Stocker la colonne par addition de 5 ml de solution de stockage (azoture de sodium à 0,02% dans du PBS). Lorsque environ 3 ml restent dans la colonne, bouchon de fond et de sécuriser le capuchon supérieur. Rangez la colonne à 4 ° C. </li><li> Déterminer la longueur oun tube de dialyse f nécessaire selon les instructions du fabricant. Utilisation de 16 mm de diamètre de tube plat utiliser 5,47 cm de longueur de tube par ml de solution à dialyse. Couper le tube et rincer avec du PBS pH 7,4 pendant 15 à 30 min. </li><li> Pliez et coupez une extrémité du tube de dialyse, transférer l&#39;IgG commun fractions contenant un tube de dialyse équilibrées dans PBS pH 7,4 et fermer l&#39;autre extrémité. </li><li> Dialyser à température ambiante contre 200 volumes de PBS, pH 7,4 pendant 2 heures. </li><li> Changez le tampon de dialyse (PBS fraîche pH 7,4) et dialyse pendant 2 h à température ambiante. </li><li> Changez le tampon de dialyse (PBS fraîche pH 7,4) et dialyse pendant la nuit à 4 ° C. </li><li> Conserver la solution d&#39;anticorps dialysée à 4 ° C jusqu&#39;au moment de procéder à d&#39;autres étapes de purification. </li><li> Évaluer la concentration d&#39;anticorps en utilisant son absorption à 280 nm et le coefficient d&#39;absorption molaire d&#39;anticorps à 280 nm de 210 000 M -1 cm -1 12. </li></ol> 2. Couplage de protéines LBD de couplage Aminolink Résine <ol><li> Préparer deux colonnes, l&#39;une pour l&#39;autre et Nor1 pour Nur77. Suivez le même protocole pour les deux protéines. </li><li> Décongeler la protéine à être couplé à la résine sur de la glace. Déterminer la concentration en protéine en utilisant le coefficient d&#39;extinction molaire et son absorbance à 280 nm 12. <ol><li> En variante, en utilisant un essai de détermination de protéine tel que le dosage de l&#39;acide bicinchoninique 13. Si la protéine est dissoute dans un tampon Dialyser ou tampon contenant une amine échange contre le tampon de couplage. Si la protéine est dans un tampon approprié (pas d&#39;amines libres) diluer 4 fois en tampon de couplage. </li></ol></li><li> Utiliser 2 ml de résine pour 2 mg de protéine. Toutes les étapes qui suivent sont fournies à résine de 2 ml dans une colonne de 10 ml. Jusqu&#39;à 10 mg de protéine peut être lié par 1 ml de résine (2 ml de 1: une suspension). </li><li> Préparer une colonne de 10 ml d&#39;une fritte en le poussant vers le bas et l&#39;exécution du PBS pH 7,4 à travers elle. Cap til fond de la colonne et ajouter 2 ml de tampon de couplage. </li><li> Ajouter le volume désiré de suspension (4 ml d&#39;obtenir 2 ml de résine) à la colonne, retirez le bouchon de fond et laisser le drain de la colonne. Rincer la colonne avec un total de 6 ml (volume 3) résine-lit de tampon de couplage et de laisser la colonne se vider. Après le tampon de couplage a drainé, remplacer le bouchon de fond. </li><li> Ajouter 2-4 ml de protéine en suspension dans de couplage de tampon à la colonne (conserver une partie aliquote de la solution de protéine afin d&#39;évaluer l&#39;efficacité de couplage par comparaison de la concentration en protéine de l&#39;éluat de l&#39;étape 2.11 en utilisant soit l&#39;absorbance à 280 nm ou le dosage bicinchoninique) . Cap et à la fin sur l&#39;extrémité mélanger pour avoir une solution homogène. </li><li> Peser un 1,8 ml microtube vide et passer à la hotte. Soigneusement transférer NaCNBH3 (environ 0,05 g) dans le tube pré-pesé. Fermer le tube et peser le tube contenant NaCNBH3. Ne pas ouvrir le tube extérieur de la hotte. </li><li> Sur la base du poids deNaCNBH3 transféré dans le tube, préparer une solution 5 M par addition de 1 M de NaOH. Le poids moléculaire du NaCNBH3 est 62,84 g donc 0,05 g est égal à (0,05 / 62,84 = 7,96 x 10 -4 mole) 7,96 x 10 -4 moles. Pour obtenir une solution add 5 M (7,96 x 10 -4 / 5) 159 pl de 1 M NaOH. </li><li> Mesurer le volume total de la réaction qui a le volume de résine en considération. Ajouter 10 pi de 5 M NaCNBH3 par ml de réaction. <ol><li> Par exemple: pour un volume de réaction de 4 ml ajouter 40 pi de 5 M NaCNBH3. Pour 1 ml de résine et 3 ml de protéine ajoutée, le volume total est de 4 ml. </li></ol></li><li> Cap de la colonne et à la fin mélanger plus fin. Fixer la colonne sur un agitateur de tube et laisser la réaction continuer pendant une nuit à 4 ° C. </li><li> Dans la matinée, porter la colonne à la hotte chimique et soigneusement enlever le bouchon comme certains gaz peut s&#39;être formée. Égoutter la colonne dans un tube propre et économiser l&#39;éluat pour mesurer la protéine concentration en utilisant l&#39;absorbance à 280 nm de 12 procédé ou le dosage de l&#39;acide bicinchoninique et le comparer avec la concentration de protéine de départ dans l&#39;étape 2.6. </li><li> Laver la résine avec 4 ml de tampon de couplage et de laisser le drain de la colonne. </li></ol> 3. Blocage sites restants <ol><li> Laver la résine avec 4 ml de tampon de trempe. Égoutter et remettre le bouchon en bas. Ajouter 2 ml de tampon de trempe et suspendre la résine. </li><li> Évaluer le volume total de réaction en mesurant le volume de la résine et le volume de tampon de trempe, puis ajouter 10 ul de 5 M NaCNBH3 par ml de volume réactionnel. </li><li> Mélanger délicatement à la température ambiante pendant 30 min fin sur la fin. Après 30 minutes, amener la colonne dans la hotte. Retirez délicatement le haut, puis retirez le bouchon de fond et laisser l&#39;évacuation de la colonne dans un tube de déchets. </li><li> Laver la colonne avec 5 volumes de résine de la solution de lavage. Surveiller les lavages de la présence de protéines résiduelles en mesurant la abso de l&#39;éluatrbance à 280 nm. Protéines non couplées sont lavés par la forte concentration en sel. </li><li> Laver la résine avec 6 ml de PBS dégazé pH 7,4 contenant de l&#39;azoture de sodium à 0,02%. Garder la colonne à 4 ° C jusqu&#39;à utilisation. </li></ol> 4. Purification de l&#39;anticorps spécifiques NURR1 <ol><li> Rincer la colonne couplé Nur77 LBD avec 10 ml de PBS pH 7,4 et laisser l&#39;évacuation de la colonne. Boucher le bas de la colonne et placer la colonne couplé Nur77 LBD drainé dans un nouveau tube de 15 ml de collecte. </li><li> Ajouter 5 ml de la protéine A des anticorps anti-NURR1 purifiés, plafonner la colonne et le placer sur un rotateur à 4 ° C pendant 1 heure. Retirez les capuchons supérieurs et inférieurs et de recueillir l&#39;écoulement à travers. Mettez de côté sur la glace jusqu&#39;à ce que prêt à ajouter à la colonne couplé Nor1 LBD. </li><li> Rincer la colonne couplé Nor1 LBD avec 10 ml de PBS pH 7,4 et laisser l&#39;évacuation de la colonne. Boucher le bas de la colonne et placer la colonne Nor1 LBD couplé à un tube de 15 ml de collecte. </li><li> Ajouter le flux à travers du coup Nur77 LBDcolonne conduite, plafonner la colonne et le placer sur un rotateur à 4 ° C pendant 1 heure. Retirez les capuchons supérieurs et inférieurs et de recueillir l&#39;écoulement à travers. </li><li> Mesurer la concentration d&#39;anticorps en mesurant l&#39;absorbance à 280 nm. </li></ol> Analyse des purifiée d&#39;anticorps anti-Nurr1 à base d&#39;ELISA 5. <ol><li> Ajouter 100 ul de protéine (2,5 pg / ml) dans les puits d&#39;une plaque à 96 puits. Si l&#39;essai 3 protéines différentes, ajouter 100 ul de protéines 1 à godets A1 à H4, 100 pi de la protéine 2, à godets A5 à H8 et 100 pi de la protéine 3 à puits A9 à H12. Couvrir la plaque et incuber une nuit à 4 ° C. </li><li> Laver deux fois les puits revêtus avec 300 pi par puits de PBS pH 7,4 et ajouter 300 ul de tampon de blocage ELISA pour les antigènes des puits revêtus entiers et incuber 2 heures à température ambiante. </li><li> Bien que la plaque bloque préparer une dilution de départ désirée de l&#39;anticorps purifié anti-Nurr1 (allant de 10 à 100 pg / ml) dans du tampon de blocage ELISA. </li><li> Après 2 h de blocage, remplacer le tampon de bloc ELISA avec 100 pi de tampon frais de bloc ELISA à tous les puits. </li><li> Ajouter 100 ul d&#39;anticorps anti-Nurr1 purifié dilué dans tous les puits dans la ligne A. diluer en série les anticorps en transférant 100 ul de solution de la ligne A dans les puits de la rangée B suivie par le transfert de 100 pi de solution de la ligne B à la ligne C continue jusqu&#39;à la ligne G. Après le mélange des solutions dans les puits dans la rangée G jeter le supplément de 100 pi. Wells dans la rangée H ne reçoivent les premiers 100 pi de tampon de blocage. Incuber à température ambiante pendant 1 heure. </li><li> Laver la plaque trois (3) fois avec 300 pi par puits de 0,05% de Tween 20 dans du PBS pH 7,4 et épongez la plaque pour éliminer le tampon de lavage en excès. </li><li> Ajouter 100 pi de la peroxydase de raifort (HRP) conjugué de chèvre anti-IgG de lapin dilué dans du tampon de bloc ELISA (1: 8000 dilution; la gamme typique est de 1: 5000 à 1: 25.000). Incuber à température ambiante pendant 1 heure. </li><li> Laver les e plaqueree (3) fois comme décrit en 5.6. Rincez tous les puits en les remplissant avec 300 pi d&#39;eau déminéralisée. Éponger l&#39;excès d&#39;eau en inversant la plaque sur du papier absorbant. </li><li> Ajouter 100 ul de 3, 3 &#39;, 5, 5&#39; tétraméthylbenzidine (réchauffé à température ambiante) à tous les puits et incuber 15 à 30 min à l&#39;obscurité à température ambiante. </li><li> Arrêter la réaction en ajoutant 50 ul de 2 NH 2 SO 4. </li><li> Lire l&#39;absorbance à 450 nm soustraction du fond à 650 nm. </li></ol>

<ol>
	<li>Hawk, J. D., Abel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+NR4A+transcription+factors+in+memory+formation.">The role of NR4A transcription factors in memory formation.</a> Brain Res. Bull. 85 (1-2), 21-29 (2011).</li><li>Law, S. W., Conneely, O. M., DeMayo, F. J., O'Malley, B. W. <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+new+brain-specific+transcription+factor,+NURR1.">Identification of a new brain-specific transcription factor, NURR1.</a> Mol. Endocrinol. 6 (12), 2129-2135 (1992).</li><li>Zetterstr&#246;m, R. 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=Dopamine+neuron+agenesis+in+Nurr1-deficient+mice.">Dopamine neuron agenesis in Nurr1-deficient mice.</a> Science. 276 (5310), 248-250 (1997).</li><li>Kadkhodaei, B., 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=Nurr1+is+required+for+maintenance+of+maturing+and+adult+midbrain+dopamine+neurons.">Nurr1 is required for maintenance of maturing and adult midbrain dopamine neurons.</a> J. Neurosci. 29 (50), 15923-15932 (2009).</li><li>Decressac, M., Volakakis, N., Bj&#246;rklund, A., Perlmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NURR1+in+Parkinson+disease--from+pathogenesis+to+therapeutic+potential.">NURR1 in Parkinson disease--from pathogenesis to therapeutic potential.</a> Nat Rev Neurol. 9 (11), 629-636 (2013).</li><li>Pe&#241;a de Ortiz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+Expression+of+the+Orphan+Nuclear+Receptor+Gene+hzf-3/nurr1+during+Spatial+Discrimination+Learning.">Hippocampal Expression of the Orphan Nuclear Receptor Gene hzf-3/nurr1 during Spatial Discrimination Learning.</a> Neurobiol Learn Mem. 74 (2), 161-175 (2000).</li><li>Vecsey, C. 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=Histone+deacetylase+inhibitors+enhance+memory+and+synaptic+plasticity+via+CREB:CBP-dependent+transcriptional+activation.">Histone deacetylase inhibitors enhance memory and synaptic plasticity via CREB:CBP-dependent transcriptional activation.</a> J. Neurosci. 27 (23), 6128-6140 (2007).</li><li>Col&#243;n-Cesario, W. I., 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=Knockdown+of+Nurr1+in+the+rat+hippocampus:+implications+to+spatial+discrimination+learning+and+memory.">Knockdown of Nurr1 in the rat hippocampus: implications to spatial discrimination learning and memory.</a> Learn Mem. 13 (6), 734-744 (2006).</li><li>McQuown, S. C., 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=HDAC3+is+a+critical+negative+regulator+of+long-term+memory+formation.">HDAC3 is a critical negative regulator of long-term memory formation.</a> J. Neurosci. 31 (2), 764-774 (2011).</li><li>Hawk, J. D., 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=NR4A+nuclear+receptors+support+memory+enhancement+by+histone+deacetylase+inhibitors.">NR4A nuclear receptors support memory enhancement by histone deacetylase inhibitors.</a> J. Clin. Invest. 122 (10), 3593-3602 (2012).</li><li>Bridi, M. S., Abel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NR4A+orphan+nuclear+receptors+mediate+transcription-dependent+hippocampal+synaptic+plasticity.">The NR4A orphan nuclear receptors mediate transcription-dependent hippocampal synaptic plasticity.</a> Neurobiol Learn Mem. 105, 151-158 (2013).</li><li>Gill, S. C., von Hippel, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+protein+extinction+coefficients+from+amino+acid+sequence+data.">Calculation of protein extinction coefficients from amino acid sequence data.</a> Anal. Biochem. 182 (2), 319-326 (1989).</li><li>Smith, P. 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=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal. Biochem. 150 (1), 76-85 (1985).</li><li>Zetterstr&#246;m, R. H., Williams, R., Perlmann, T., Olson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+expression+of+the+immediate+early+transcription+factors+Nurr1+and+NGFI-B+suggests+a+gene+regulatory+role+in+several+brain+regions+including+the+nigrostriatal+dopamine+system.">Cellular expression of the immediate early transcription factors Nurr1 and NGFI-B suggests a gene regulatory role in several brain regions including the nigrostriatal dopamine system.</a> Brain Res. Mol. Brain Res. 41 (1-2), 111-120 (1996).</li><li>Xiao, Q., Castillo, S. O., Nikodem, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+messenger+RNAs+for+the+orphan+nuclear+receptors+Nurr1+and+Nur77+(NGFI-B)+in+adult+rat+brain+using+in+situ+hybridization.">Distribution of messenger RNAs for the orphan nuclear receptors Nurr1 and Nur77 (NGFI-B) in adult rat brain using in situ hybridization.</a> Neuroscience. 75 (1), 221-230 (1996).</li></ol>

Les auteurs ont rien à révéler.

Le succès de ce protocole repose sur la disponibilité de protéines pures pour la caractérisation des anticorps dirigés contre un membre spécifique de la famille de la protéine d&#39;intérêt. Il n&#39;y a aucune nécessité de purifier les anticorps à l&#39;aide de protéine A / G jusqu&#39;à ce qu&#39;il soit clairement établi qu&#39;il existe des réactivités croisées significatives par ELISA et Western blot. Comme il est indiqué dans le texte, il est important d&#39;éviter ...

Le facteur de transcription Nurr1 et ses homologues (Nur77 et NOR1) appartiennent à la sous-famille des récepteurs nucléaires 4A (NR4A) 1. Ils sont également des récepteurs orphelins parce que leurs ligands endogènes ne sont pas encore identifiés. Nurr1 a été cloné en 1992 et bien connu pour être exprimé dans le cerveau 2, son rôle essentiel pour le développement et la maintenance des neurones dopaminergiques du mésencéphale a été révélé par des études de souris knockout 3. En outre, son rôle important pour le maintien des neurones dopaminergiques du mésencéphale a récemment été démontré par une étude de knock-out conditionnel 4. En raison de ces études montrent élégantes rôles critiques de NURR1 pour les neurones dopaminergiques du mésencéphale, de nombreuses études ultérieures ont essentiellement porté sur la neurones dopaminergiques du mésencéphale et le trouble neurodégénératif liés à la dopamine, la maladie de Parkinson (PD) 5. Notamment, Nurr1 est exprimée non seulement dans les dopamine mésencéphale (MDA) neurones, mais aussi dans les diles zones du cerveau verset 2, suggérant que cela peut avoir des rôles fonctionnels dans de nombreux domaines non-Da, ce qui est fortement soutenue par des études plus récentes montrent que Nurr1 joue des rôles importants dans diverses fonctions du cerveau. Pour des exemples, il a été montré que les activités de mémoire induisant comme l&#39;apprentissage, ou d&#39;autres tâches de dépendante de l&#39;hippocampe résultat dans la régulation de l&#39;expression Nurr1 dans l&#39;hippocampe 6,7. En outre, abattre d&#39;expression Nurr1 dans l&#39;hippocampe était suffisante pour altérer la mémoire à long terme et / ou la plasticité synaptique 8-11, suggérant fortement que Nurr1 joue divers rôles dans de nombreuses zones du cerveau. Ainsi, afin de mieux comprendre l&#39;expression de type cellule-spécifique et subcellulaire de Nurr1, il est souhaitable d&#39;utiliser des anticorps spécifiques de NURR1, qui ne présentent pas de réactivité croisée à ses homologues Nur77 ou NOR1. Ce document décrit un protocole pré-adsorption pour générer des anticorps spécifiques NURR1 et de présenter des données supplémentaires montrant sa spécificité.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Purified Ligand Binding Domain from Nurr1, Nor 1 and Nur77</td>
			<td></td>
			<td></td>
			<td>Column Storage solution</td>
		</tr>
		<tr>
			<td>AminoLink Coupling Resin and Kit</td>
			<td>Thermo Scientific</td>
			<td>44890</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein A spin column</td>
			<td>Thermo Scientific</td>
			<td>89978</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well Flate-bottom plates, High Flange, 330&#956;L</td>
			<td>Thermo Scientific</td>
			<td>3455</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Normal Goat Serum</td>
			<td>KPL</td>
			<td>50-675-69</td>
			<td></td>
		</tr>
		<tr>
			<td>Milk Diluent/ Blocking Concentrate&#160;</td>
			<td>KPL</td>
			<td>50-82-01</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG Elution Buffer</td>
			<td>Thermo Scientific</td>
			<td>21004</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 1 M Tris-HCI Buffer, pH 7.5</td>
			<td>Life Technologies</td>
			<td>15567-&#173;&#8208;027</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Radish Peroxidase conjugated Goat anti-Rabbit IgG (H+L)</td>
			<td>Thermo Scientific</td>
			<td>31460</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Resin</td>
			<td>Thermo Scientific</td>
			<td>88221</td>
			<td></td>
		</tr>
		<tr>
			<td>3, 3&#39;, 5, 5&#39; Tetramethylbenzydine</td>
			<td>Thermo Scientific</td>
			<td>34028</td>
			<td></td>
		</tr>
		<tr>
			<td>2N H2SO4</td>
			<td>Macron Fine Chemicals</td>
			<td>H381 05</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein A IgG Binding Buffer</td>
			<td>Thermo Scientific</td>
			<td>21001</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG Elution Buffer</td>
			<td>Thermo Scientific</td>
			<td>21004</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutralization Buffer</td>
			<td></td>
			<td></td>
			<td>1M Tris-&#173;&#8208;HCl pH 8.5</td>
		</tr>
		<tr>
			<td>Column Storage solution</td>
			<td></td>
			<td></td>
			<td>Phosphate Buffered Saline containing 0.02% sodium azide</td>
		</tr>
		<tr>
			<td>Spectra/Por 7 pre-treated dialysis tubing</td>
			<td>Spectrum Labs</td>
			<td>132128</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml Disposable columns</td>
			<td>Thermo Scientific</td>
			<td>29924</td>
			<td></td>
		</tr>
		<tr>
			<td>AminoLink Coupling Buffer</td>
			<td></td>
			<td></td>
			<td>0.1M sodium phosphate, 0.05% NaN3, pH 7.0</td>
		</tr>
		<tr>
			<td>Quenching buffer</td>
			<td></td>
			<td></td>
			<td>1M Tris. HCI, 0.05% NaN3, pH 
			7.4</td>
		</tr>
		<tr>
			<td>Wash Solution</td>
			<td></td>
			<td></td>
			<td>1M NaCl, 0.05% NaN3</td>
		</tr>
		<tr>
			<td>ELISA Blocking buffer</td>
			<td></td>
			<td></td>
			<td>1% Normal Horse serum (NHS), 1% Normal Goat Serum (NGS) in 1X KPL milk diluent</td>
		</tr>
		<tr>
			<td>Storage Solution</td>
			<td></td>
			<td></td>
			<td>PBS pH 7.4 containing 0.02% sodium azide</td>
		</tr>
		<tr>
			<td>Micropipettes: 10 &#956;l; 20 &#956;l; 200 &#956;l; 1000 &#956;l</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

production of nurr-1 specific polyclonal antibodies free of cross-reactivity against its close homologs, nor1 and nur77

The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The nuclear receptor related 1 protein (Nurr1 or NR4A2) plays a key role in the maintenance of the dopaminergic system. Dopamine dysfunctions associated with the Nurr1 gene include Parkinson&#8217;s disease, schizophrenia and manic depression among others. Furthermore, recent evidence indicates that Nurr1 is also expressed in other brain areas such as the hippocampus and plays critical roles for learning and memory. The other members of the family are nerve growth factor IB (Nur77 or NR4A1) and neuron-derived orphan receptor 1 (NOR1 or NR4A3). To help investigate the precise functional roles of Nurr1 in dopaminergic and other brain region-related neuronal dysfunctions antibodies devoid of cross-reactivities against Nur77 and NOR1 were needed. Since the proteins are more divergent in their LBDs than in their DNA binding domains immunization with purified LBDs should yield antibodies specific for Nurr1 with minimal reactivities against Nur77 and/or NOR1. Although anti-Nurr1 antibodies were successfully generated these showed significant immunoreactivity against the other members of the family. Affinity chromatography over immobilized Protein A followed by pre-adsorption against immobilized Nur77 and NOR1 LBDs yielded Nurr1 specific antibodies free of cross-reactivity. Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption. The goal of the protocol is to increase polyclonal antibodies specificity through pre-adsorption against cross-reactive antigens.

Une comparaison de colonne de protéine A purifiée d&#39;anticorps spécifiques de NURR1 avec la protéine A des anticorps purifiés anti-NURR1 suivis par le passage à travers Nur77 LBD et Nor1 LBD colonnes est illustrée sur la figure 1. Comme on peut le constater, la protéine A purifiée anticorps Nurr1 montré une forte liaison à Nurr1 LBD. Toutefois, il a également montré une liaison significative à Nur77 LBD et Nor1 LBD. Lorsque la protéine d&#39;anticorps A purifié NURR1 ont été encore ...

Watch this Scientific Journal Video about Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77 at JoVE.com

Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77

Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption.

Production de Nurr-1 anticorps polyclonaux spécifiques gratuit de réactivité croisée contre son étroite homologues, Nor1 et Nur77

The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The ...

production-nurr-1-specific-polyclonal-antibodies-free-cross

Research

JoVE Journal

Biology

7.4K vues.  McLean Hospital. Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption.

Vidéo: Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77

Operant Learning of Drosophila at the Torque Meter

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized breeding regime assures proper larval density and age-matched cohorts. Cold-anesthetized flies are glued with head and thorax to a triangle-shaped hook the day before the experiment. Attached to the torque meter via a clamp, the fly's intended flight maneuvers are measured as the angular momentum around its vertical body axis. The fly is placed in the center of a cylindrical panorama to accomplish stationary flight. An analog to digital converter card feeds the yaw torque signal into a computer which stores the trace for later analysis. The computer also controls a variety of stimuli which can be brought under the fly's control by closing the feedback loop between these stimuli and the yaw torque trace. Punishment is achieved by applying heat from an adjustable infrared laser.

Measuring the yaw torque of tethered Drosophila with the torque meter allows the neuroscientist exquisite control of the stimulus situation of the experimental animal. Together with the unique genetic tools available in the fruit fly, this paradigm is used for a wide variety of neurobiological research.

L&#39;apprentissage opérant de la drosophile à la couplemètre

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized ...

Recherche

Biologie

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

Démonstration technique de l&#39;hybridation tableau Whole Genome génomique comparative

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

In vivo Ca 2 + - Imagerie des neurones du corps champignons Pendant l&#39;apprentissage olfactif dans le Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Cellules de mammifères autonome bioluminescentes pour la surveillance continue et en temps réel de la cytotoxicité

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Conception et analyse du comportement de préférence de température et de son rythme circadien chez la drosophile

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

Incorporation spécifiques résidus de noncanonical acides aminés en protéines de modèle à l&#39;aide d&#39;un<em&gt; Escherichia coli</em&gt; Système de transcription-traduction libre Cell-

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is phosphorylated by both receptor and non-receptor tyrosine kinases at tyrosine 1798. Omori et al. developed site- and phosphorylation status-specific antibodies against human girdin at tyrosine-1798 (pY1798), which specifically bind to phosphorylated tyrosine-1798, but not to unphosphorylated tyrosine-1798. pY1798 antibodies have been used to specifically label tuft cells (TCs) that are present in mammalian gastrointestinal tissues, but the function of these cells is unclear. This protocol allows the robust visualization of TCs in the jejunum using pY1798 antibodies and immunofluorescence. To ensure successful and simple TC visualization, this protocol includes two histological techniques: production of free-floating cryosections from gelatin-filled jejunum tissue, and low-temperature antigen retrieval at 50 &#176;C for 3 h. Filling the jejunum with gelatin maintains the shape of free-floating sections throughout the staining procedure, whereas low-temperature antigen retrieval ensures robust signals from TCs. Successful use of this protocol results in pY1798 staining of TCs distributed from villus tip to crypt. Stained TCs have a spool-shaped soma and fluorescent signals condense at the lumenal tip, which corresponds to the protruding &#39;tuft.&#39;&#160;Phalloidin staining colocalized with pY1798-positive TCs at the thickened brush border, and corresponds to a rootlet mass extending from the TC tuft. This protocol could be used to examine TCs in human biopsy samples collected with gastrointestinal endoscopes. Furthermore, TCs were recently reported to accumulate following parasite infection in mice, suggesting that this protocol could have applications for diagnosis of parasite infections in the human gut.

Kuga et al. discovered that phosphorylation-status specific antibodies against the actin binding protein girdin phosphorylated at tyrosine 1798 (pY1798) can be used to label tuft cells (TCs). This protocol allows robust visualization of TCs using immunofluorescent staining of free-floating jejunum cryosections with pY1798 antibodies.

Utilisation des anticorps Anti-phospho-girdin pour visualiser la touffe Intestinal cellules de souris flottante jéjunum Cryosections

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

Agrobacterium-Transformation génétique médiée, production transgénique et application pour l’étude du développement de la reproduction masculine dans le riz

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are significantly different among different regions of seed. In order to view the morphologies of cells, starch granules and protein bodies clearly, and quantitatively analyze their morphology parameters accurately, the whole-seed-sized section is needed. Though the whole-seed-sized paraffin section can investigate the accumulation of storage materials in seeds, it is very difficult to quantitatively analyze the morphology parameters of cells and storage materials due to the low resolution of the thick section. The thin resin section has high resolution, but the routine resin sectioning method is not suitable to prepare the whole-seed-sized section of mature seeds with a large volume and high starch content. In this study, we present a simple dry sectioning method for preparing the whole-seed-sized resin section. The technique can prepare the cross and longitudinal whole-seed-sized sections of developing, mature, germinated, and cooked seeds embedded in LR White resin, even for large seeds with high starch content. The whole-seed-sized section can be stained with fluorescent brightener 28, iodine, and Coomassie brilliant blue R250 to specifically exhibit the morphology of cells, starch granules, and protein bodies clearly, respectively. The image obtained can also be analyzed quantitatively to show the morphology parameters of cells, starch granules, and protein bodies in different regions of seed.

This technique allows for the fast and simple preparation of whole-seed-sized resin section for the observation and analysis of cells, starch granules, and protein bodies in different regions of the seed.

Une méthode simple de section à sec pour l’obtention de la section résine de la taille d’une graine entière et de ses applications

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are ...

Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate

Cell-free gene expression offers the power of biology without the complications of a living organism. Although many such gene expression systems exist, most are quite expensive to buy and/or require special equipment and finely honed expertise to produce effectively. This protocol describes a method to produce bacterial cell-free lysate that supports high levels of gene expression, using only standard laboratory equipment and requiring minimal processing. The method uses an Escherichia coli strain producing an endolysin that does not affect growth, but which efficiently lyses a harvested cell pellet following a simple freeze-thaw cycle. The only further processing required is a brief incubation followed by centrifugation to clear the autolysate of cellular debris. Dynamic gene circuits can be achieved through heterologous expression of the ClpX protease in the cells before harvesting. An E. coli strain lacking the lacZ gene can be used for high-sensitivity, cell-free biosensing applications using a colorimetric or fluorescent readout. The entire protocol requires as few as 8-9 hours, with only 1-2 hours of hands-on labor from inoculation to completion. By reducing the cost and time to obtain cell-free lysate, this method should increase the affordability of cell-free gene expression for various applications.

This protocol describes a rapid and simple method to produce bacterial lysate for cell-free gene expression, using an engineered strain of Escherichia coli and requiring only standard laboratory equipment.

Production rapide, abordable et simple de lysat sans cellules bactériennes

Cell-free gene expression offers the power of biology without the complications of a living organism. Although many such gene expression systems exist, ...