Name: production of nurr-1 specific polyclonal antibodies free of cross-reactivity against its close homologs, nor1 and nur77
Uploaded: 2015-08-17T14:00:00
Description: 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

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

Примечание: состав всех решений, приведенных ниже, можно найти в материалах / Оборудование таблице. 1. Белок Колонка антител Очистка <ol><li> Равновесие белка в колонку отжима и все необходимые буферы до комнатной температуры в течение 15 мин до начала процедуры. </li><li> Развес...

<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>

Успех этого протокола зависит от наличия чистых белков для характеризации антител, против конкретного члена семейства белков интереса. Там нет необходимости, чтобы очистить антител с использованием белка A / G, пока не будет четко установлено, что существуют значительные перекрестных ?...

Транскрипционный фактор Nurr1 и его гомологов (Nur77 и Nor1) относятся к ядерной рецепторов подсемейства 4А (NR4A) 1. Они также сироты рецепторы, потому что их эндогенные лиганды пока не определены. Nurr1 впервые клонировали в 1992 году, и, хотя, как известно, выражается в мозге 2, его существенную роль для развития и поддержания среднего мозга нейронов допамина было выявлено в исследованиях нокаутом мыши 3. Кроме того, его важная роль для поддержания среднего мозга нейронов допамина недавно была продемонстрирована в исследовании условного нокаута 4. Из-за этих исследований, показывающих, элегантные решающую роль Nurr1 для среднего мозга нейронов допамина, многие последующие исследования в основном сосредоточены на среднем мозге нейроны допамина и допамина нейродегенеративных расстройств, болезни Паркинсона (БП) 5. Следует отметить, что Nurr1 выражается не только в нейронах среднего мозга допамина (МДА), но также и в диучастки мозга стих 2, предполагая, что он может иметь функциональные роли во многих областях, не-DA, который сильно поддерживается более недавних исследований, показывающих, что Nurr1 играет важную роль в различных функций мозга. Для примера, было показано, что память вызывающие действия, такие как обучение, или другие гиппокамп-зависимые задачи приводит к повышающей регуляции экспрессии Nurr1 в гиппокампе 6,7. Кроме того, сбить из Nurr1 выражения в гиппокампе было достаточно, чтобы нарушить долгосрочную память и / или синаптической пластичности 8-11, сильно предполагая, что Nurr1 играет различные роли во многих областях головного мозга. Таким образом, для дальнейшего понимания типов клеток специфический и внутриклеточную экспрессию Nurr1, желательно использовать Nurr1-специфические антитела, которые не обладают какой-либо перекрестной реактивности с его гомологи или Nur77 Nor1. Эта статья описывает протокол предварительного адсорбции генерировать Nurr1-специфические антитела и представить дополнительные данные, показывающие, свою специфику.

<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.

Сравнение белка колонку очищают Nurr1-специфические антитела с белком A очищенные антитела анти-Nurr1 затем прохождения через Nur77 ДТЛ и Nor1 LBD колонн, показанные на рисунке 1. Как видно, белок очищали Nurr1 антитела выставлены сильной связи чтобы Nurr1 ДТЛ. Тем не менее, он также показал зна...

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.

Производство Nurr-1 Удельная поликлональных антител Бесплатные перекрестной реактивности против своих Близкие гомологи, Nor1 и 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 ...

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