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

Not: Aşağıda belirtilen tüm çözümlerin kompozisyon Malzemeleri / Ekipman Tablo bulunabilir. 1. Protein A Sütun Antikor Arıtma <ol><li> Bir protein A spin kolon ve önceki işleme başlamadan 15 dakika boyunca oda sıcaklığına kadar gerekli tüm tamponları dengelenmesi. </li><li> Bağışıklık serumu 1 seyreltin: 1 Protein A IgG Bağlama Tamponu ile. (Serum 5 mi tavsiye edilir). </li><li> Yavaşça kapağı açılmış olabilecek reçineyi çıkarmak için bir proteine ​​tezgah üstünde bir spin kolon dokunun. Üst kapağı çıkarın ve yavaşça alt kapatma koparmak. 15 ml toplama tüpüne sütun yerleştirin ve depolama çözümü süzülmesini bekleyin. </li><li> Bağlama Tamponu Protein A IgG 5 ml ilave edilir ve çözelti, kurumasına izin verin. </li><li> Sütuna seyreltilmiş bağışıklık serumu uygulayın ve flow-through toplamak. En iyi sonuçlar için, sütunun antikor-bağlanma capacit% 80 daha az bir antikor konsantrasyonunu içeren bir örnek hacim eklemeky. </li><li> Bağlama Tamponu Protein A IgG, 15 ml ya da 280 nm&#39;de absorbans aşağıdaki 0.1 kadar sütun yıkayın. </li><li> Beş etiketli toplama tüplerine Nötralizasyon Tamponu 100 ul ekleyin. </li><li> Protein A sütun IgG Elüsyon Tamponu 5 ml ilave edilir ve Nötralizasyon tampon 100 ul ihtiva eden tüplerin her biri, 1 ml&#39;lik fraksiyonların toplanması. </li><li> 0.5&#39;den daha büyük olan bir absorbans 280 nm ve havuz fraksiyonlar her fraksiyondan absorbansı ölçülür. Diyaliz için hazır olana kadar buz üzerinde Bir araya toplanan fraksiyonlar tutun. </li><li> IgG Elüsyon Tamponu 8 ml ekleyerek sütun yeniden oluşturun ve çözelti sütun üzerinden akmasına izin verir. </li><li> 7.4&#39;e yıkama sıvısı, pH dönene kadar Protein A IgG bağlanma solüsyonu ile sütun durulayın. </li><li> Depolama çözeltisi 5 ml (PBS içinde% 0.02 sodyum azid) eklenerek sütun saklayın. Yaklaşık 3 ml sütun, kapak altındaki kalır ve üst kapağı emniyete zaman. 4 ° C &#39;de sütun saklayın. </li><li> Uzunluk o belirleyinf Diyaliz tübü, üreticinin talimatlarına uygun olarak gerekli. 16 mm, düz çaplı boru çözeltinin ml&#39;si başına boru uzunluğunun 5.47 cm kullanımı kullanılarak diyaliz edilmesi. Boru kesme ve 15-30 dakika boyunca PBS pH 7.4 ile yıkayın. </li><li> Katlama ve diyaliz hortumun bir ucunu klip, PBS pH 7.4 içinde dengelenmiş diyaliz tüp içeren fraksiyonlar bir araya toplanmış IgG aktarmak ve diğer ucu kapatın. </li><li> 2 saat süre ile, PBS, pH 7.4, 200 hacmine karşı oda sıcaklığında Dialyze. </li><li> Diyaliz tamponu (taze PBS pH 7.4) değiştirin ve oda sıcaklığında başka bir 2 saat boyunca diyaliz. </li><li> Diyaliz tamponu (taze PBS pH 7.4) değiştirin ve 4 ° C&#39;de gece boyunca diyaliz. </li><li> Daha fazla saflaştırma adımları ile devam etmek için hazır olana kadar 4 ° C&#39;de diyaliz edilir antikor çözüm tutun. </li><li> 280 nm&#39;de absorbans kullanılarak antikor konsantrasyonuna ve 210,000 M-1 cm-1 12 ve 280 nm &#39;de antikor Molar soğurma katsayısı değerlendirin. </li></ol> LBD Proteinlerin 2. Bağlantı Bağlantı Reçine AminoLink için <ol><li> Iki sütun, Nor1 diğeri Nur77 diğer hazırlayın. Her iki protein için de aynı protokol takip edin. </li><li> Protein çözülme buz üzerinde reçineye bağlanmak üzere. Onun moler sönüm katsayısı ve 280 mil 12, emiciliğinin kullanarak protein konsantrasyonunu belirleyin. <ol><li> Alternatif olarak, bu bisinkoninik asit yöntemi 13 gibi bir protein tayini testi kullanılır. Protein bağlama tamponuna karşı bir amin-içeren tampon diyaliz ya da tampon alışverişi içinde eritildi edin. Protein, Uygun bir tampon maddesi içinde (serbest aminler) ise bu, bağlayıcı tampon içinde 4 kat seyreltilir. </li></ol></li><li> Protein 2 mg reçine 2 ml kullanın. Izleyen tüm adımlar 10 mi sütununda 2 mi reçine içindir. Protein 10 mg kadar reçine, 1 ml (1: bulamacın 1 2 ml) başına bağlanabilir. </li><li> Altına bir cam hamuru bastırıyor ve içinden PBS pH 7.4 çalıştırarak bir 10 ml sütun hazırlayın. Cap tO kolonun alt ve Kaplin Tampon 2 ml ekleyin. </li><li> , Kolona (reçine 2 ml elde etmek için 4 ml) çamurun istenen hacim ekleme alt kapağı çıkarın ve sütun drenaj sağlar. Bağlayıcı tampon içinde 6 ml (3 reçine yatak hacmi) &#39;in bir toplam sütun durulayın ve sütun boşalmasını sağlar. Kavrama tampon süzüldükten sonra, alt kapağı değiştirin. </li><li> (280 nm&#39;de emicilik ya bisinkoninik deneyi kullanarak adım 2.11 elüatın protein konsantrasyonu karşılaştırarak bağlanma verimliliğini değerlendirmek için protein çözeltisi bir kısım devam) kolonuna bağlayıcı tampon içinde süspansiyon haline getirilmiş protein, 2-4 ml ekleyin . Cap ve homojen bir çözüm var uca üzerine sonunu karıştırın. </li><li> Boş 1.8 ml&#39;lik ependorf tüp tartılır ve davlumbaz taşımak. Dikkatli bir şekilde önceden tartılmış bir tüp içinde NaCNBH3 (yaklaşık 0.05 g) aktarın. Tüp kapatılır ve NaCNBH3 içeren tüp tartın. Kaput dışında tüp açmayın. </li><li> Ağırlığına göreNaCNBH3, tüpüne aktarıldı, 1 M NaOH ilave edilerek 5 M bir solüsyon yapmak. NaCNBH3 moleküler ağırlığı 62.84 gr, bu nedenle 0.05 g (0.05 / 62.84 = 7.96 x 10 -4 mol) 7.96 x 10 -4 mol eşit olmasıdır. 5 M solüsyonu (7.96 x 10 -4/5), 1 M NaOH ile 159 ul yapmak için. </li><li> Reaksiyonun toplam hacmi dikkate reçine hacmi alarak ölçün. Reaksiyonun, ml başına 5 M NaCNBH3 10 ul ekle. <ol><li> Örnek: Bir 4 ml reaksiyon hacmi için 5 M NaCNBH3 40 ul ekle. Reçine 1 ml ilave protein, 3 ml için, toplam hacim 4 ml&#39;dir. </li></ol></li><li> Sütun Cap ve sonunda üzerinde sonunu karıştırın. Bir tüp döndürücü sütun Güvenli ve reaksiyon 4 ° C&#39;de gece boyunca devam edelim. </li><li> Sabah, kimyasal kaput sütun getirmek ve dikkatli bir gaz oluşmuş olabilir gibi kapağı çıkarın. Temiz bir tüp içine sütun boşaltın ve protein con ölçmek için eluatı kaydetmek280 nm yöntemiyle 12 absorbans veya bisinkoninik asit deneyi ve adım 2.6 başlayarak protein konsantrasyonu ile karşılaştırmak kullanarak santrasyonu. </li><li> Kavrama 4 ml tampon ile reçinenin yıkanır ve sütun süzdürülür. </li></ol> 3. Engelleme Kalan Siteler <ol><li> Tampon söndürülmesi 4 ml reçine yıkanır. Drenaj ve alt kapağını kapatın. Tampon söndürülmesi 2 ml ekleyin ve reçine askıya. </li><li> Reçine hacmi ve söndürme tampon hacmi daha sonra reaksiyon hacmi ml başına 5 M NaCNBH3 10 ul ekleyin ölçerek toplam reaksiyon hacmi değerlendirin. </li><li> 30 dakika ucu-üzerinde-sonu boyunca oda sıcaklığında yavaşça karıştırın. 30 dakika sonra, davlumbaz sütun getir. Dikkatle üst kaldırmak ve daha sonra alt kapağı çıkarın ve bir atık tüp içine sütun tahliye edelim. </li><li> Yıkama çözeltisi 5 reçine hacimleri ile sütun yıkayın. Taşıma ürününden en abso ölçülmesiyle bakiye proteinlerinin varlığı için yıkama Monitör280 nm&#39;de rbance. Kuplajsız proteinler yüksek tuz konsantrasyonu ile yıkanır. </li><li> Gazı alınmış, PBS, pH 7.4,% 0.02 sodyum azid, 6 ml reçine yıkanır. Gerekli olana kadar, 4 ° C &#39;de sütun tutun. </li></ol> Nurr1 Özgü Antikorlar 4. saflaştırılması <ol><li> PBS pH 7.4 10 ml Nur77 LBD bağlanmış sütun durulayın ve sütun süzdürülür. Sütunun alt Cap ve yeni bir 15 ml toplama tüpü drene Nur77 LBD birleştiğinde sütun yerleştirin. </li><li> Protein 5 ml saflaştırılmış bir anti-Nurr1 antikor ekleyin 1 saat boyunca 4 ° C &#39;de, bir döndürücü üzerinde sütun ve yer kap. Üst ve alt kapaklarını çıkarın ve akışı toplamak. Nor1 LBD birleştiğinde sütuna eklemek için hazır olana kadar buz üzerinde bir kenara koyun. </li><li> PBS pH 7.4 10 ml Nor1 LBD bağlanmış sütun durulayın ve sütun süzdürülür. Sütunun alt kapağı ve bir 15 ml&#39;lik bir toplama tüpü içinde Nor1 LBD bağlanmış sütun yerleştirin. </li><li> Nur77 LBD darbeden akışını yoluyla Ekleyol kolon, 1 saat boyunca 4 ° C &#39;de, bir döndürücü üzerinde sütun ve yer kap. Üst ve alt kapaklarını çıkarın ve akışı toplamak. </li><li> 280 nm&#39;de absorbans ölçümü ile antikor konsantrasyonu ölçümü. </li></ol> Saf Anti-Nurr1 Antikor 5. ELISA-tabanlı Analiz <ol><li> 96 oyuklu bir plakanın oyuklarına protein, 100 ul (2.5 ug / ml) ilave et. 3 farklı protein test için, H4 Wells A1, H8 Wells A5 proteinin 2, 100 ul, ve H12 kuyu A9 proteinin 3 100 ul proteinin 1. 100 ul ilave edin. Plaka Kapak ve 4 ° C&#39;de bir gece inkübe edin. </li><li> PBS pH 7.4, oyuk başına 300 ul iki kez kaplanmış oyuklarına yıkayın ve tüm antijen kaplı oyuklara ELISA Bloke edici tamponun 300 ul ilave edildi ve oda sıcaklığında 2 saat inkübe et. </li><li> Plakası saflaştırılmış anti-Nurr1 antikorun arzu edilen çıkış seyreltme hazırlanması bloke edilirken ELISA bloke tamponu içinde (10 ile 100 ug / ml arasında değişen). </li><li> Engelleme 2 saat sonra, tüm oyuklara taze ELISA blok tamponunda 100 ul ELISA blok tamponu değiştirin. </li><li> Seri olarak C satır satır B çözeltisinin 100 ul aktarma ardından satır B çukurlara satır A çözeltisi 100 ul aktarma ile antikorları seyreltik satır A&#39;da tüm oyuklara seyreltildi saflaştırılmış anti-Nurr1 antikoru 100 ul ekle aşağı doğru devam G ekstra 100 ul atmak aralıksız kuyularda çözüm karıştırıldıktan sonra G. satır. Satır H Wells, sadece tampon engelleme ilk 100 ul alırsınız. 1 saat oda sıcaklığında inkübe edin. </li><li> PBS pH 7.4 plaka,% 0,05 Tween 20, oyuk başına 300 ul olan üç (3) defa yıkayın ve fazla yıkama tamponu çıkması için plakayı blot. </li><li> Yaban turpu peroksidazı (HRP), ELISA Blok tamponu içinde seyreltilmiş biyotinle konjuge edilmiş keçi anti-tavşan IgG, 100 ul ekle (1: 8000 seyreltme, tipik bir aralığı, 1: 25,000: 5,000 ile 1 arası). 1 saat oda sıcaklığında inkübe edin. </li><li> Plaka th yıkayınREE (3) defa 5.6 de tarif edildiği gibi. Iyonu giderilmiş su, 300 ul ile doldurarak her kuyu durulayın. Emici kağıt üzerine plakayı baş aşağı çevirerek fazla suyu kurulayın. </li><li> Tüm oyuklara tetrametilbenzidin (oda sıcaklığına kadar ılıtıldı) 3 100 ul, 3 &#39;5, 5&#39;, ilave edilir ve oda sıcaklığında 15 karanlıkta 30 dakika için inkübe edilir. </li><li> SO 4 NH2 2 50 ul ilave ederek reaksiyonu durdurun. </li><li> 650 nm &#39;de bir arka plan çıkarılması 450 nm&#39;de absorbansı okumak. </li></ol>

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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>

Yazarlar ifşa hiçbir şey yok.

Bu protokolün bir başarı ilgili protein ailesinin belirli bir üye karşı üretilen antikorların karakterizasyonu için, saf proteinler mevcudiyetine dayanır. Bu açık bir şekilde ELISA ve Western blot analizi ile önemli çapraz reaktivite olduğu kurulana kadar protein A / G kullanılarak antikorları saflaştırmak için bir ihtiyaç vardır. Metinde belirtildiği gibi, bu tür Tris ya da glisin gibi bir amin-ihtiva eden tampon maddesi içinde sütuna çapraz bağlanmış olduğu ...

Transkripsiyon faktörü Nurr1 ve homologları (Nur77 ve Nor1), nükleer reseptör alt ailesi 4A (NR4A) 1 aittir. Onların endojen ligandlar henüz tespit edilmemiş çünkü onlar da yetim reseptörler vardır. Nurr1 ilk olarak 1992 klonlandı ve bilinen, ancak beyin 2&#39;de belirtildiği üzere, geliştirme ve orta beyin dopamin nöronlarının bakımı için de önemli bir rolü nakavt fare çalışmaları 3 konuldu. Buna ek olarak, orta beyin dopamin nöronlarının bakım için çok önemli bir rol son şartlı bir nakavt çalışma 4 ile gösterilmiştir. Nedeniyle orta beyin dopamin nöronları için Nurr1 en kritik roller gösteren bu zarif çalışmalara, birçok sonraki çalışmalarda büyük ölçüde dopamin nöronları ve dopamin ile ilişkili nörodejeneratif bozukluk orta beyin odaklanmıştır, Parkinson hastalığı (PH) 5. Özellikle, Nurr1 orta beyin dopamin (MDA) nöronlarda, aynı zamanda di sadece ifade edilirşiddetle Nurr1 çeşitli beyin fonksiyonlarında önemli rol oynadığını gösteren daha yeni çalışmalar tarafından desteklenmektedir olmayan birçok DA alanlarda işlevsel rollere sahip olabileceğini düşündüren ayet beyin bölgelerinin 2. Örnekler için, bu gibi öğrenme gibi bellek indükleyici aktiviteleri gösterilir, ya da diğer hipokampus bağımlı görevler yukarı regülasyonu hipokampus 6,7 Nurr1 ifade ile sonuçlanır. Buna ek olarak, aşağı hipokampus Nurr1 ifade vurmak şiddetle Nurr1 birçok beyin bölgelerindeki çeşitli roller oynadığını düşündüren, uzun süreli hafızayı ve / veya sinaptik plastisite 8-11 zarar için yeterli oldu. Bu nedenle, daha Nurr1 hücre-tipi-spesifik ve hücre içi ifade anlamak için, onun homologları ya da Nur77 Nor1 herhangi bir çapraz reaktivite sergilemez Nurr1 özgü antikorları kullanmak için tercih edilir. Bu kağıt özgüllüğü gösteren Nurr1 özgü antikorlar ve bu ek verileri oluşturmak için bir ön-adsorpsiyonu protokolü tarif eder.

<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.

Protein bir karşılaştırması Nur77 LBD ve Nor1 LBD sütunları boyunca geçirilerek ve ardından bir saflaştırılmış anti-Nurr1 antikorları görülebilir Şekil 1. As gösterilmiştir, Protein A sütun saf hale Nurr1-spesifik antikorlar, protein-A saf hale Nurr1 antikor güçlü bir bağlanma sergilemiştir Nurr1 LBD&#39;sine. Bununla birlikte, aynı zamanda Nur77 LBD ve Nor1 LBD bağlanmasını belirgin göstermiştir. Protein A saflaştırılmış Nurr1 antikorlar ayrıca Nur77 LBD ve Nor1 LB...

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.

Bu yakın homologları, Nor1 ve Nur77 karşı çapraz-reaktivite Nurr-1 spesifik olan poliklonal antikorlar Free üretimi

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 Görüntüleme.  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.

Video: 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.

Site Özel Recombinase (SSR) Teknoloji İlkeleri

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

Biyoloji

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.

Tork Metre Drosophila Edimsel Öğrenme

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 + - Bal Arısı Koku Öğrenme sırasında Mantar Vücut Nöronlar Görüntüleme

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.

Sitotoksisite Sürekli ve gerçek zamanlı izlenmesi için özerk Bioluminescent Memeli Hücre

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.

Drosophila'da Sıcaklık Tercih Davranışının ve Sirkadiyen Ritminin Tasarımı ve Analizi

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

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

Floresan etiketli Nanopartiküller kullanma Fare, İnsan ve domuz Menşe Canlı hücreler hücre içi Gen İfade Algılama

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

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.

Bir Kullanarak Modeli Proteinler içine Kuralsız Amino Asit kalıntı özgü Ortaklığın<em&gt; Escherichia coli</em&gt; Hücre içermeyen Transkripsiyon-çeviri Sistemi

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.

Serbest dalgalanma fare değişir Cryosections hücrelerde bağırsak tutam görselleştirmek için Anti-fosfo-girdin antikor kullanımı

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-Aracılı Genetik Dönüşüm, Transgenik Üretim ve Pirinçte Erkek Üreme Gelişimi Çalışmaları Için Uygulaması

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.