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

Nota: A composição de todas as soluções a seguir indicadas podem ser encontrados na Tabela Materiais / Equipamento. 1. Proteína A coluna de anticorpo Purificação <ol><li> Equilibrar a coluna de proteína A rotação e todos os tampões necessários à temperatura ambiente durante 15 min antes de iniciar o procedimento. </li><li> Diluir o soro imune 1: 1 com a proteína A de IgG Binding Buffer. (5 ml de soro é recomendado). </li><li> Bata levemente uma coluna de proteína rodada na bancada para desalojar qualquer resina que pode ser apresentado na tampa. Remova a tampa superior e encaixe delicadamente fora do fecho da parte inferior. Inserir a coluna no tubo de recolha de 15 ml de solução de armazenamento e permitir o seu escoamento. </li><li> Adicionar 5 ml de Proteína A IgG tampão de ligação e permitir que a solução escorra. </li><li> Aplicar o soro imune diluído para a coluna e recolher o fluxo de passagem. Para melhores resultados, adicionar um volume de amostra que contém uma concentração de anticorpos menos de 80% da ligação do anticorpo-capacit da colunay. </li><li> Lava-se a coluna com 15 ml de Proteína A IgG Binding Buffer ou até a absorvância a 280 nm é inferior a 0,1. </li><li> Adicione 100 ml de Neutralização Buffer para cinco tubos de coleta marcadas. </li><li> Adicionar 5 ml de tampão de eluição de IgG ao da Proteína A coluna e recolher fracções de 1 ml em cada um dos tubos contendo 100 ul de tampão de neutralização. </li><li> Medir a absorção de cada fração de 280 fracções nm e piscina com uma absorvância superior a 0,5. Mantenha as fracções reunidas em gelo até estar pronto para diálise. </li><li> Regenerar a coluna por adição de 8 ml de tampão de eluição de IgG e permitir que a solução flui através da coluna. </li><li> Lavar a coluna com solução de ligação a IgG da Proteína A, até os retornos eluente de pH para 7,4. </li><li> Armazenar a coluna através da adição de 5 ml de solução de armazenamento (azida de sódio a 0,02% em PBS). Quando cerca de 3 ml permanecem na coluna, tampa inferior e prenda a tampa superior. Armazenar a coluna a 4 ° C. </li><li> Determinar o comprimento Of tubagem de diálise necessária de acordo com as instruções do fabricante. Usando tubagem plana 16 milímetros de diâmetro utilizar 5,47 centímetro de comprimento do tubo por ml de solução a ser dialisado. Cortar o tubo e enxaguar com PBS pH 7,4 durante 15 a 30 min. </li><li> Dobrar e cortar uma extremidade do tubo de diálise, a transferência de IgG reunida continha as fracções para tubo de diálise equilibrada em PBS pH 7,4 e fechar a outra extremidade. </li><li> Dialisar à temperatura ambiente contra 200 volumes de PBS, pH 7,4 durante 2 horas. </li><li> Mudar o tampão de diálise (PBS fresco pH 7,4) e dializar durante mais 2 horas à temperatura ambiente. </li><li> Mudar o tampão de diálise (PBS fresco pH 7,4) e diálise durante a noite a 4 ° C. </li><li> Manter a solução de anticorpo dialisado a 4 ° C até estar pronto para prosseguir com outros passos de purificação. </li><li> Avaliar a concentração de anticorpo utilizando a sua absorvância a 280 nm, e o coeficiente de absorção molar de anticorpos a 280 nm de 210,000 M-1 cm-1 12. </li></ol> 2. Acoplamento de proteínas LBD para Aminolink Resina Acoplamento <ol><li> Prepare duas colunas, uma para a outra e NOR1 para Nur77. Seguir o mesmo protocolo para ambas as proteínas. </li><li> Descongelar a proteína a ser acoplada à resina em gelo. Determinar a concentração de proteína utilizando o seu coeficiente de extinção molar e a sua absorvância a 280 nm de 12. <ol><li> Em alternativa, usar um ensaio de determinação de proteínas, tais como o ensaio de ácido bicinconínico 13. Se a proteína é dissolvida num tampão de diálise de troca contendo amina ou tampão contra a atrelagem. Se a proteína é de um tampão adequado (não há aminas livres) dilua 4 vezes em tampão de acoplamento. </li></ol></li><li> Utilizar 2 ml de resina por 2 mg de proteína. Todos os passos que se seguem são para a resina de 2 ml numa coluna de 10 ml. Até 10 mg de proteína pode ser ligado por 1 ml de resina (2 ml de 1: 1) pasta. </li><li> Prepara-se uma coluna de 10 ml por meio de uma frita de empurrar para o fundo e rodando PBS pH 7,4 através dele. Cap tele parte inferior da coluna e adicionar 2 ml de tampão de acoplamento. </li><li> Adicionar o volume pretendido de suspensão (4 ml para se obter 2 ml de resina) para a coluna, remover a tampa de fundo e deixar a coluna de drenagem. Lavar a coluna com um total de 6 ml (volume de leito de resina 3) de atrelagem e permite que a coluna para drenar. Depois que o tampão de acoplamento tenha escorrido, recoloque a tampa inferior. </li><li> Adicionar 2-4 ml de proteína suspensa em tampão de acoplamento para a coluna (manter-se uma alíquota da solução de proteína para avaliar a eficiência de acoplamento por comparação da concentração de proteína do eluato na etapa 2.11 usando a absorvância a 280 nm ou o ensaio de ácido bicinconínico) . Tampa e misture sobre a extremidade final para ter uma solução homogénea. </li><li> Pesar um tubo de microcentrífuga de 1,8 ml vazio e passar para a capela. Transferir cuidadosamente NaCNBH3 (cerca de 0,05 g) no tubo pré-pesado. Fechar o tubo e pesar o tubo contendo NaCNBH3. Não abra o tubo fora do capô. </li><li> Com base no peso deNaCNBH3 transferida para o tubo, fazer uma solução 5 M por adição de NaOH 1 M. O peso molecular de NaCNBH3, por conseguinte, é 62,84 g 0,05 g é igual a (0,05 / 62,84 = 7,96 x 10 -4 mol) 7,96 x 10 -4 mol. Para fazer uma solução 5 M de suplemento (7,96 x 10 -4 / 5) 159 ul de NaOH 1 M. </li><li> Medir o volume total da reacção de tomar o volume de resina em consideração. Adicionar 10 ul de 5 M NaCNBH3 por ml de reacção. <ol><li> Por exemplo: para um volume de reacção 4 ml de adicionar 40 ul de 5 M de NaCNBH3. Para 1 mL de resina e 3 ml de proteína adicionado, o volume total é de 4 ml. </li></ol></li><li> Tampe a coluna e misturar sobre a extremidade final. Fixar a coluna num rotor tubo e deixar a reacção prosseguir durante a noite a 4 ° C. </li><li> Na parte da manhã, trazer a coluna para a capa química e retire cuidadosamente a tampa como um pouco de gás pode ter se formado. Drenar a coluna para um tubo limpo e guardar o eluato para medir a proteína concentração usando a absorvância a 280 nm, método 12 ou o ensaio de ácido bicinconínico e compará-la com a concentração de proteínas a partir de passo 2.6. </li><li> Lavou-se a resina com 4 ml de tampão de acoplamento e deixar a coluna de drenagem. </li></ol> 3. Bloqueio restante Sites <ol><li> Lavou-se a resina com 4 ml de tampão de têmpera. Escorra e coloque a tampa inferior. Adicionar 2 ml de tampão de têmpera e suspender a resina. </li><li> Avaliar o volume total da reacção por medição do volume de resina e do volume de tampão de têmpera, em seguida, adicionar 10 ul de 5 M de NaCNBH3 por ml de volume de reacção. </li><li> Misturar suavemente à temperatura ambiente durante 30 min-extremidade sobre extremidade. Depois de 30 minutos, trazer a coluna no exaustor de fumos. Remova cuidadosamente a parte superior e, em seguida, retire a tampa do fundo e deixar o dreno coluna em um tubo de resíduos. </li><li> Lavar a coluna com 5 volumes de resina de solução de lavagem. Monitorizar as lavagens para a presença de proteínas residuais medindo abso do eluatorbance a 280 nm. Proteínas desacoplados são lavados para fora pela concentração elevada de sal. </li><li> Lavou-se a resina com 6 ml de PBS desgaseificado pH 7,4 contendo azida de sódio a 0,02%. Manter a coluna a 4 ° C até ser necessário. </li></ol> 4. Purificação de anticorpos específicos Nurr1 <ol><li> Lavar a coluna acoplada Nur77 LBD com 10 ml de PBS pH 7,4 e deixa a coluna de drenagem. Tapar o fundo da coluna e colocar a coluna acoplada Nur77 LBD drenada em 15 ml de um novo tubo de recolha. </li><li> Adicionam-se 5 ml de proteína A anticorpos anti-Nurr1 purificados, tampão da coluna e lugar num agitador rotativo a 4 ° C durante 1 h. Remova as tampas superior e inferior e recolher o fluxo através. Separe em gelo até estar pronto para adicionar à coluna acoplada NOR1 LBD. </li><li> Lavar a coluna acoplada NOR1 LBD com 10 ml de PBS pH 7,4 e deixa a coluna de drenagem. Tapar o fundo da coluna e colocar o NOR1 LBD coluna acoplada em um tubo de recolha de 15 ml. </li><li> Adicionar o fluxo através do golpe Nur77 LBDlevou coluna, tampão da coluna e lugar num agitador rotativo a 4 ° C durante 1 hora. Remova as tampas superior e inferior e recolher o fluxo através. </li><li> Medir a concentração de anticorpos através da medição da absorvância a 280 nm. </li></ol> Análise baseada em ELISA de 5. purificada Anticorpo Anti-Nurr1 <ol><li> Adicionar 100 ul de proteína (2,5 ug / ml) aos poços de uma placa de 96 poços. Se testar três diferentes proteínas, adicione 100 ml de proteína de 1 a poços A1 a H4, 100 l de proteína 2, a Wells A5 para H8 e 100 l de proteína 3 a poços A9 para H12. Cobrir a placa e incubar durante a noite a 4 ° C. </li><li> Lavar os poços revestidos duas vezes com 300 ul por poço de PBS, pH 7,4 e adicionar 300 ul de tampão de bloqueio de ELISA para os antigénios inteiros poços revestidos e incubar 2 horas à temperatura ambiente. </li><li> Enquanto o prato está a bloquear preparar uma diluição de partida desejado purificado do anticorpo anti-Nurr1 (variando de 10 a 100 ug / ml) em tampão de bloqueamento de ELISA. </li><li> Após 2 horas de bloqueio, substitua o buffer do bloco de ELISA com 100 ul de tampão de bloqueio fresco ELISA para todos os poços. </li><li> Adiciona-se 100 ul de anticorpo purificado anti-Nurr1 diluída a todos os poços em linha em série A. diluir os anticorpos por transferência de 100 ul de solução da linha A para a linha B em poços seguidos por transferência de 100 ul de solução de linha B para a linha C continuando até a linha G. Depois de misturar as soluções em poços na fileira G descartar as extra de 100 mL. Wells em linha H só recebem os primeiros 100 ml de tampão de bloqueio. Incubar à temperatura ambiente durante 1 h. </li><li> Lava-se a placa três (3) vezes com 300 ul por cavidade de 0,05% de Tween 20 em PBS pH 7,4 e seque a placa para remover o excesso de tampão de lavagem. </li><li> Adicionar 100 ul de peroxidase de rábano (HRP) conjugado de cabra anti-IgG de coelho diluído em tampão de bloqueio de ELISA (1: 8000 de diluição, a gama típica é de 1: 5000 a 1: 25.000). Incubar à temperatura ambiente durante 1 h. </li><li> Lavar as th placaree (3) vezes, como descrito em 5.6. Lave todos os poços, preenchendo-os com 300 mL de água deionizada. Remova o excesso de água invertendo a placa sobre papel absorvente. </li><li> Adicionar 100 ul de 3, 3 &#39;, 5, 5&#39; tetrametilbenzidina (aquecida até à temperatura ambiente) a todos os poços e incubar 15 a 30 min no escuro à temperatura ambiente. </li><li> Parar a reacção por adição de 50 ul de 2 NH 2 SO 4. </li><li> Ler a absorvância a 450 nm subtraindo fundo a 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>

Os autores não têm nada a revelar.

O sucesso deste protocolo baseia-se na disponibilidade de proteínas puras para a caracterização dos anticorpos criados contra um membro específico da família de proteínas de interesse. Não há necessidade de purificar os anticorpos utilizando proteína A / G até que seja claramente estabelecido que existem significativas reacções cruzadas por ELISA e Western blot. Conforme indicado no texto, é importante para evitar a suspensão de proteína (s) a ser reticulado à coluna num tamp...

O factor de transcrição e os seus homólogos de Nurr1 (Nur77 e NOR1) pertencem à subfamília de receptores nucleares 4A (NR4A) 1. Eles também são receptores órfãos porque seus ligantes endógenos ainda não foram identificados. Nurr1 foi primeiro clonado em 1992 e, embora conhecido por ser expresso no cérebro 2, o seu papel essencial para o desenvolvimento e manutenção de neurónios dopaminérgicos do mesencéfalo foi revelado por estudos de ratinho knockout 3. Além disso, o seu importante papel para a manutenção dos neurônios de dopamina do mesencéfalo foi recentemente demonstrado por um estudo nocaute condicional 4. Devido a estes estudos elegantes mostrando papéis críticos do Nurr1 para os neurônios de dopamina do mesencéfalo, muitos estudos subsequentes em grande parte focado no neurônios de dopamina e de doenças neurodegenerativas relacionadas com a dopamina do mesencéfalo, doença de Parkinson (DP) 5. Notavelmente, Nurr1 se expressa não apenas nos neurônios de dopamina do mesencéfalo (MDA), mas também em diáreas cerebrais verso 2, sugerindo que ele pode ter papéis funcionais em muitas áreas não-Da, que é fortemente apoiada por estudos mais recentes que mostram que Nurr1 desempenha papéis importantes em várias funções cerebrais. Por exemplo, demonstrou-se que as actividades de memória induzir tal como a aprendizagem, ou outras tarefas hipocampo-dependentes resultar em aumento da regulação da expressão no hipocampo Nurr1 6,7. Além disso, derrubar Nurr1 de expressão no hipocampo foi suficiente para prejudicar a memória a longo prazo e / ou a plasticidade sináptica 8-11, sugerindo fortemente que Nurr1 desempenha diversos papéis em muitas áreas do cérebro. Assim, para melhor compreender a expressão específica do tipo celular e subcelular de Nurr1, é desejável a utilização de anticorpos específicos de Nurr1, que não apresentam qualquer reactividade cruzada com os seus homólogos Nur77 ou NOR1. Este documento descreve um protocolo de pré-adsorção para gerar anticorpos específicos de Nurr1 e apresentar dados adicionais mostrando a sua especificidade.

<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.

Uma comparação da coluna de proteína A purificada anticorpos específicos para a proteína A Nurr1 com anticorpos anti-Nurr1 purificados seguido por passagem através de colunas Nur77 LBD e NOR1 LBD é mostrado na Figura 1. Tal como pode ser visto, Proteína-A purificada Nurr1 anticorpo exibiram uma forte ligação para Nurr1 LBD. No entanto, também mostraram ligação significativa a Nur77 LBD e NOR1 LBD. Quando os anticorpos Nurr1-A proteína purificada foram ainda purificados contra Nur77 LBD e N...

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.

Produção de Nurr-1 policlonal específico Anticorpos Livre de reactividade cruzada contra o seu próximo homólogos, NOR1 e 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 visualizações.  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.

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

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

Princípios do site-specific Recombinase (SSR) Tecnologia

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Biologia

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.

Aprendizagem operante de Drosophila no medidor de Torque

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 ...

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 + - Imagem de Neurônios Corpo Mushroom durante o aprendizado olfativo no 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.

Células de mamíferos autonomamente bioluminescentes para monitoramento contínuo e em tempo real de citotoxicidade

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.

Projeto e Análise do Comportamento de Preferência de Temperatura e seu Ritmo Circadiano em Drosophila

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.

Incorporação específico de resíduos de não-canônicos Aminoácidos em proteínas modelo Usando um<em&gt; Escherichia coli</em&gt; Sistema de transcrição-tradução livre-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.

Uso de anticorpos Antifosfo-girdin Visualizar células intestinais topete no Mouse flutuante 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 ...

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-Transformação Genética Mediada, Produção Transgênica e Sua Aplicação para o Estudo do Desenvolvimento Reprodutivo Masculino no Arroz

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.

Um método simples de secção seca para obter seção de resina de tamanho inteiro e suas aplicações

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.

Produção rápida, acessível e descomplicada de lysate sem células bacterianas

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