Bu eser hibe Bakanlığı bilim ve Teknoloji (PCC için çoğu, 105-2320-B-010-007-MY3), Ulusal Sağlık Araştırma Enstitüsü (NHRI-EX105-10215BC için PCC), Bakanlığı bilim ve Teknoloji (en 105-2314-B-400-019 tarafından desteklenmiştir HJK için) ve Ulusal Sağlık Araştırma Enstitüsü (NHRI MG-105-SP-10, NHRI MG-106-SP-10 HJK için). Bu eser de kısmen Ulusal Yang Ming Üniversitesi Tarih PCC yayınına el yazması ile desteklenmiştir. Fon çalışma tasarım, veri toplama ve analizi, yayımlamaya karar veya tazminat makalenin herhangi bir rolü yoktu.

1. hazırlanması Baculovirus ifade oluşturur
<ol><li>
Bir çift ifade baculovirus vektör K-bZIP19 cDNA klonlama tarafından SUMO E3 ligaz K-bZIP arındırmak (tablo malzemeleri görmek) bir N-terminal epitope-etiketi ile. (İletişim kuralı olarak vektör-etiket-K-bZIP boyunca gösterilen) octapeptide etiketini kullanarak bir başarı olmuştur. Not: K-bZIP Polimeraz zincir reaksiyonu (PCR) klonlama DNA cDNA ters gelen Doksisiklin tedavi20takip TREx F3H3-K-Rta BCBL-1 hücrelerden izole RNA transkripsiyonu şablonudur.<ol>
<li>Tam uzunlukta K-bZIP cDNA19 ben siteleri klonlama Cpoile çift ifade vektör içine aktarın. CPO PCR ürünü ve çift ifade vektör 3 h2137 ° C'de 1 µg sindirmek.</li>
		<li>Elektroforez bir % 0,8 özel jel22tarihinde takip sindirilir DNA ayıklamak ve ayrı. Ekle (K-bZIP) ligate ve vektör T4 DNA ligaz için üreticinin yönergelerine uygun 30 dk 16 ° C'de tarafından (bkz. Tablo malzeme).</li>
		<li>5 µL eklemek yetkili E. coli ligasyonu DNA için 50 µL olan bir 1,5 mL tüp içinde "A" (bakınız Tablo reçetesi) hücreleri. 30 dk için buza koy. Sonra bir 42 ° C su banyosunda 45 için 1,5 mL tüp kuluçkaya s ve hemen 3 dakika buzda Tüpü yerleştirmek.</li>
		<li>600 µL S.O.C. orta (%0,5 (w/v) Maya ekstresi, %2 (w/v) tryptone, 10 mM NaCl, 2.5 mM KCl, 20 mM MgSO4ve % 4 (w/v) glikoz) tüpün içine ekleyin ve 1 h için 37 ° C'de kuluçkaya. O zaman, tüp 600 x g de 3 dakika oda sıcaklığında santrifüj kapasitesi.</li>
		<li>Süpernatant kaldırmak, pelet 60 µL Luria-Bertani orta ile resuspend (LB; %1 (w/v) tryptone, % 0,5 (w/v) Maya özü ve 85 mM NaCl) ve 100 µg/mL Ampisilin içeren LB agar plakaları yaymak. Agar plaka 16 h23,2437 ° C'de kuluçkaya.</li>
		<li>5 mL LB orta 100 µg/mL Ampisilin ve kültür için 16 h 37 ° C'de 250 sallayarak d / içeren aşılamak için 1 koloni seçin. Sonra bir plazmid ekstraksiyon kiti üreticinin yönergelerine uygun olarak plazmid, vektör-etiket-K-bZIP, özü ( Tablo malzemelerigörmek).</li>
	</ol>
</li>
	<li>
Yetkili E. coli içine vektör-etiket-K-bZIP dönüşüm tarafından etiket-K-bZIP yataklık rekombinant bacmid oluşturmak (bkz. tablo reçetesi) hücreleri "B".
	<ol>
<li>Mix 0.2 µg vektör-etiket-K-bZIP Plazmid ve 100 µL yetkili E. coli hücreleri "B" bir 1,5 mL tüp ve 30 dakika süreyle koymak üzerinde buz kuluçkaya tüp 42 ° C su banyosunda 45 için s ve hemen 3 dakika buzda Tüpü yerleştirmek.</li>
		<li>S.O.C. orta 900 µL tüpün içine ekleyin ve 50 RPM döndürme için 4 h 37 ° C'de kuluçkaya. Daha sonra orta 10 µL al, S.O.C orta 50 µg/mL sefaloridin, 7 µg/mL gentamisin, 10 µg/mL tetrasiklin, 100 µg/mL galaktozidaz substrat ve 40 µg/mL izopropil β-D-1-thiogalactopyranoside (IPTG içeren LB agar Tabaklarda yaymak için ek bir 50 µL Ekle ) ve tabaklar 37 ° C'de 48 h için kuluçkaya</li>
		<li>5 mL LB orta 50 µg/mL sefaloridin, 7 µg/mL gentamisin ve 10 µg/mL tetrasiklin içeren bir beyaz kolonisi aşılamak. 250 için 16 h sallayarak rpm ile 37 ° C'de kuluçkaya.</li>
		<li>Rekombinant bacmid etiket-K-bZIP barındıran DNA izole etmek, ölçmek ve 1 µg/µL25bir konsantrasyon yeniden askıya alma.</li>
	</ol>
</li>
	<li>
Rekombinant baculoviruses etiket-K-bZIP rekombinant bacmid 6-şey plaka bir iyi Sf9 hücrelere etiket-K-bZIP içeren DNA transfecting 1 µg tarafından ifade oluşturur.
	<ol>
<li>(Bkz. Tablo reçetesi) tohum 2 x 106 Sf9 hücrelerde 2 mL Grace'in böcek % 10 fetal sığır serum (FBS) ve her %1 gentamisin ile birlikte orta bir 6 iyi tabak o zaman için 1 h 27 ° C'de kuluçkaya.</li>
		<li>Grace'in böcek %10 FBS, % 1 gentamisin ve % 1 deterjan C ile birlikte orta hücrelerde Sf9 günlük faz kültürünü korumak ( Tablo malzemelerigörmek) 140 devirde yörünge süspansiyon 27 ° C'de. Bir hemasitometre ve trypan mavi boyama kullanarak hücreleri saymak; hücre canlılığı % 95 fazla olmamalıdır. (En az yoğunluk ~0.5 x 106 hücre/mL) 2-3 günde bir subcultivation oranı 1:3 kullanarak hücreleri korumak.</li>
		<li>2 µL bacmid DNA ekleyin (1 µg/µL) ve 98 µL düşük serum medya (bkz. Tablo reçetesi) 2 mL tüp içine. Daha sonra 4 µL transfection reaktif ekleyin ( Tablo malzemelerigörmek) tüp ve mix iyi. 15 dakika oda sıcaklığında kuluçkaya.</li>
		<li>Bu transfection karışımı (104 µL) her kuyunun içine ekleyin ve 12-16 saat 27 ° C'de kuluçkaya.</li>
		<li>Nazik aspirasyon ile yorgun medyayı çıkarın ve 2 mL taze Grace'in böcek % 10 FBS ve % 1 gentamisin ile sağlanan orta yerine. Sf9 hücreler gevşek plaka yüzeyine uygun ve nazik aspirasyon ile rahatsız edilmek istemiyorum.</li>
	</ol>
</li>
	<li>
Rekombinant baculovirus transfection sonra toplamak ve yüksek-titresi stokları (P1) daha fazla deneyler için elde etmek için baculovirus yükseltmek.
	<ol>
<li>Orta, scrape Sf9 hücreleri steril hücre lifter kullanarak değiştirdikten sonra 72 h toplamak süpernatant 1,5 mL tüp ve girdap 10 için tek tek her kuyudan s.</li>
		<li>150 x g., 3 dk 4 ° C'de toplamak süpernatant P0 baculovirus ve aliquot 1,5 mL tüpler (her tüpün içinde 1 mL baculovirus) olarak santrifüj kapasitesi. Mağaza-80 ° C'de</li>
		<li>Tohum 1 x 107 Sf9 10 mL Grace'in böcek orta hücrelerde 10 cm Petri kabına %10 FBS ve % 1 gentamisin ile sağlanan ve 1 h için 27 ° C'de kuluçkaya.</li>
		<li>Rekombinant baculoviruses ifade tag-K-bZIP amplifikasyon, 0.5 mL P0 baculovirus numaralı seribaşı Sf9 hücrelere ekleme ve 48 h 27 ° C'de kuluçkaya.</li>
		<li>Süpernatant P1 baculovirus 15 mL tüp ve store 4 ° c ilâ 1 ay boyunca toplamak gibi.</li>
	</ol>
</li>
</ol>2. K-bZIP saflaştırılması
<ol><li>Grace'in böcek 1.3.2. adımda açıklandığı gibi %10 FBS, % 1 gentamisin ve 140 devirde yörünge süspansiyon 27 ° C'de % 1 deterjan C ile birlikte orta hücrelerde Sf9 günlük faz kültürünü korumak.</li>
	<li>İletim günü, yoğunluklu olarak 2 x 106 hücre/mL 250 mL Sf9 hücre içine baculovirus içeren süpernatant (P1) 1 mL ekleyin.</li>
	<li>Sf9 hücreler için bir orbital Çalkalayıcı üzerinde 27 ° C'de 72 h 140 sallayarak d / kuluçkaya sonra Santrifüjü 2,740 x g 4 ° C'de 15 dakika de tarafından toplamak</li>
	<li>Süpernatant kaldırmak ve hücre topakları içinde 10 mL lizis tampon (20 mM HEPES pH 7.9, 0.5 M NaCl, % 1 deterjan (bakınız Tablo malzemeler), % 2 gliserol, proteaz inhibitörü kokteyl) koşullar ve Döndür'ün 50 RPM bir süspansiyon karıştırıcı 4 ° C'de 30 dk için kullanma.</li>
	<li>Santrifüj hücre süpernatant toplamak 15 dak için 15.000 x g de lysate.</li>
	<li>Etiket-K-bZIP arındırmak için hücre lysates (10 mL) ile antikor öğesini manyetik boncuklar 14 mL polipropilen borular içinde 50 µL kuluçkaya ve süspansiyon Mikser kullanarak 4 ° C'de 3 h için 50 RPM döndürün. Protein yakalama, takip cips boncuk 800 g aralıklarla 30 x 4 ° C'de s Boncuk yaklaşık 1 mL kalan hacmi yeniden askıya alma ve 1,5 mL tüp yıkama için transfer süpernatant çoğunu çıkarın.</li>
	<li>Manyetik bir stand üstündeki içeren tüp yerleştirerek yakalanan protein yıkama ve manyetik boncuklar tamamen tüp tarafına yapıştırılır bir kez süpernatant kaldırın.</li>
	<li>
Boncuk lizis arabelleği ile dört kez yıkayın.
	<ol>
<li>1 mL lizis arabellek 1,5 mL tüpler içine ekleyin ve tüpler 10 kez tersine çevirin. 1.5 mL tüp manyetik bir stand üzerine yerleştirin ve süpernatant 2.7 içinde konusunda açıklanan şekilde kaldırın.</li>
		<li>Önceki adımı başka bir 3 kez tekrarlayın.</li>
	</ol>
</li>
	<li>
Boncuk fosfat tamponlu tuz (PBS) ile bir kez yıkayın.
	<ol>
<li>1 mL PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4) 1,5 mL tüpler içine ekleyin ve tüpler 10 kez tersine çevirin.</li>
		<li>1.5 mL tüp manyetik bir mahkemeye ve süpernatant kaldır.</li>
	</ol>
</li>
	<li>Antikor öğesini manyetik boncuklar etiket-K-bZIP protein 1.5 mL tüp içine PBS içinde seyreltilmiş 150 µg/mL octapeptide 100 µL ekleyerek elute. Süspansiyon karıştırıcı oda sıcaklığında tarafından 10 dk 50 RPM için tüp döndürün. O zaman, tüp manyetik kürsüye yerleştirin ve K bZIP içeren süpernatant temiz 1.5 mL tüp içinde toplamak.</li>
	<li>1-5 saf µL etiket-K-bZIP protein SDS-sayfa26boyama Coomassie mavi tarafından takip tarafından analiz. Yük 2 µg, 1 µg ve sığır serum albumin (BSA) aynı 0,5 µg örnekleri jel ve ImageJ gibi bir görüntü işleme programında miktar için kullanabilirsiniz. K-bZIP konsantrasyon kıyasla BSA standartlara uygun tahmin ediyoruz.</li>
	<li>Arıtılmış K bZIP artık her SUMOylation assay olarak kullanılmak üzere hazırdır. K-bZIP PBS içinde 100 ng/µL ve küçük aliquots-80 ° C'de deposunda son bir konsantrasyon sulandırmak</li>
</ol>3. SUMOylation tahlil
<ol><li>100 ng/µL arıtılmış etiket-K-bZIP 3 µL her vitro SUMOylation reaksiyon ana karışımı içine ekleyin. Ana karışımında bileşenleri içeren 1 µL protein arabellek, 1 SUMOylation arabellek, 0.5 µL p53 proteini (0,5 mg/mL), 25 x enzimi aktive nM E1 (hisse senedi konsantrasyonlu: 1 µM), 50 enzim conjugating nM E2 (hisse senedi konsantrasyonlu: 10 µM) ve 250 her SUMO peptidler (SUMO-1 nM , SUMO-2 veya SUMO-3; hisse senedi konsantrasyonlu: 5 mikron) 17 µL son hacmi için.</li>
	<li>İçeriği hafifçe karıştırın ve 2 SDS-sayfa yükleme arabellek x 20 µL ekleyerek tepki 3 h. durdurmak için 30 ° C'de tepki kuluçkaya (100 mM Tris-HCL pH 6.8, % 4 Sodyum Lauryl Sülfat, %0,2 (w/v) bromophenol mavi, %20 (v/v) gliserol ve 200 mM β-mercaptoethanol) ve 95 ° c 5 min için örnekleri denatüre.</li>
	<li>
SDS-sayfasında, ayrı örnekleri elektroforetik transfer yarı kuru cihazlar kullanarak ayrılmış proteinlerin PVDF membran üzerine aktarmak ve Western blot tarafından anti-p53 antikor kullanarak analiz.
	<ol>
<li>Jel aparatı adım 2.11 olduğu gibi ayarlayın ve örnek 20 µL jel kuyu yükleyin.</li>
		<li>(Alt jel boya bant ulaşıncaya kadar) % 10 jel yaklaşık 120 dk 80 V geçerli bir sabit çalıştırın. Elektroforez tamamlanmasından sonra güç kaynağını kapatmak.</li>
		<li>Jel aparatı ve jel çekmek için jel kaset kesin ve jel yarı kuru Aktarım arabellek (48 mM Tris-HCl, 39 mM glisin, % 20 metanol) 5 min için içine float.</li>
		<li>Başka bir kabı alın ve PVDF membran metanol için 1 dk içinde ıslatın. Sonra PVDF metanol dışarı almak ve yarı kuru Aktarım arabellek koydu. Yavaşça membran 5 min için tahrik.</li>
		<li>Yarı kuru elektroforetik cihaz Emanet kapağını çıkarın.</li>
		<li>Önceden filtre kağıdı ıslak ve jel alt Platin anot üzerinde aşağıdaki gibi sandviç: filtre kağıdı, PVDF membran, jel ve filtre kağıdı.</li>
		<li>O zaman leke 15 V sabit 90 dk. çevirmek için geçerli güç kaynağını, koşmak güvenli katot plaka ve güvenlik kapağı, yarı kuru cihazlar bağlantısını kesin ve PVDF membran almak.</li>
		<li>Blok PVDF membran ile engelleme arabellek (%5 yağsız süt TBST tampon (137 mM NaCl2, 20 mM Tris-HCl, % 0,1 deterjan B (bakınız Tablo malzemeler), pH 7,6)), oda sıcaklığında 30 tarafından orbital Çalkalayıcı sallayarak devir/dakika ile 1 saat için.</li>
		<li>PVDF membran ile seyreltilmiş anti-p53 antikor (1:1, 000) 12-16 saat 4 ° C'de için engelleme arabellekte bir süspansiyon Mikser kullanarak 30 rpm sallayarak ile melez.</li>
		<li>PVDF membran bir kaba alıp TBST içinde koy. PVDF membran TBST iki kez daha fazla ile yıkayın.</li>
		<li>PVDF membran TBST içinde 45 RPM tarafından orbital Çalkalayıcı sallayarak için 30 dk bekletin.</li>
		<li>PVDF membran seyreltilmiş horseradish peroksidaz ile (HRP) Birleşik Anti-tavşan antikor ile (1:4, 000) 30 bir süspansiyon Mikser kullanarak sallayarak d / oda sıcaklığında 1 h için engelleme arabellekte melezlemek.</li>
		<li>PVDF membran 3.4.10 adımda anlatıldığı gibi TBST 3 kez ile yıkayın.</li>
		<li>PVDF membran TBST içinde 45 rpm ile sallayarak bir süspansiyon Mikser kullanarak için 30 dk bekletin. TBST kaldırın ve PVDF membran en fazla 12 saat için 4 ° C'de korumak için PBS ekleyin.</li>
		<li>Gelişmiş chemiluminescent substrat (ECL substrat) Reaktif 1 ve 2 (1:1) ( Tablo malzemelerigörmek) karıştırın. PVDF membran PBS kaldırmak, kısaca bir kağıt havlu veya aşırı nem emme ve hemen eklemek 400 µL ECL reaktif 3-5 dk. Kaldır aşırı ECL reaktif için her membran yüzeyine kısaca Blot tarafından ışık görev silecek ile leke ama m izin verme embrane için tamamen kuru.</li>
		<li>Bir ışıldama görüntüleme sistemi veya autoradiography film27kullanarak leke ortaya çıkarmak.</li>
	</ol>
</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+E2-mediated+SUMO+conjugation+revealed+by+a+complex+between+ubiquitin-conjugating+enzyme+Ubc9+and+RanGAP1.">Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1.</a> Cell. 108 (3), 345-356 (2002).</li><li>Knipscheer, P., 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=Ubc9+sumoylation+regulates+SUMO+target+discrimination.">Ubc9 sumoylation regulates SUMO target discrimination.</a> Mol Cell. 31 (3), 371-382 (2008).</li><li>Vethantham, V., Manley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+sumoylation+of+recombinant+proteins+and+subsequent+purification+for+use+in+enzymatic+assays.">In vitro sumoylation of recombinant proteins and subsequent purification for use in enzymatic assays.</a> Cold Spring Harb Protoc. 2009 (1), 5121 (2009).</li><li>Lin, S. F., Robinson, D. R., Miller, G., Kung, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposi's+sarcoma-associated+herpesvirus+encodes+a+bZIP+protein+with+homology+to+BZLF1+of+Epstein-Barr+virus.">Kaposi's sarcoma-associated herpesvirus encodes a bZIP protein with homology to BZLF1 of Epstein-Barr virus.</a> J Virol. 73 (3), 1909-1917 (1999).</li><li>Yang, W. S., Hsu, H. W., Campbell, M., Cheng, C. Y., Chang, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=K-bZIP+Mediated+SUMO-2/3+Specific+Modification+on+the+KSHV+Genome+Negatively+Regulates+Lytic+Gene+Expression+and+Viral+Reactivation.">K-bZIP Mediated SUMO-2/3 Specific Modification on the KSHV Genome Negatively Regulates Lytic Gene Expression and Viral Reactivation.</a> PLoS Pathog. 11 (7), 1005051 (2015).</li><li>Connor, C. D., Metcalf, E., Wrighton, C. J., Harris, T. J., Saunders, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RsrII--a+novel+restriction+endonuclease+with+a+heptanucleotide+recognition+site.">RsrII--a novel restriction endonuclease with a heptanucleotide recognition site.</a> Nucleic Acids Res. 12 (17), 6701-6708 (1984).</li><li>Helling, R. B., Goodman, H. M., Boyer, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+endonuclease+R-EcoRI+fragments+of+DNA+from+lambdoid+bacteriophages+and+other+viruses+by+agarose-gel+electrophoresis.">Analysis of endonuclease R-EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis.</a> J Virol. 14 (5), 1235-1244 (1974).</li><li>Sezonov, G., Joseleau-Petit, D., D'Ari, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+physiology+in+Luria-Bertani+broth.">Escherichia coli physiology in Luria-Bertani broth.</a> J Bacteriol. 189 (23), 8746-8749 (2007).</li><li>Gingold, E. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Ahmed, S., Holt, M., Riedel, D., Jahn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-scale+isolation+of+synaptic+vesicles+from+mammalian+brain.">Small-scale isolation of synaptic vesicles from mammalian brain.</a> Nat Protoc. 8 (5), 998-1009 (2013).</li></ol>

Yazarlar hiçbir potansiyel çıkar çatışmaları ifşa.

Vitro burada açıklanan SUMOylation Protokolü rutin olarak tanımlanan Ubc9 yüzeylerde SUMOylation durumunu kurmak için kullanılır. SUMO E3 ligaz SUMOylation işlevinin çalışması için standart protokolü kullanarak büyük kısıtlamadır SUMO E1 etkinleştirme ve SUMO konjugasyon vitro sistemlerinde en yüksek düzeye E2 conjugating enzimler Ubc9 bereket. Bu meydan okuma göz önüne alındığında, bu titrasyon düzeye SUMO E1 ve E2 enzim miktarının bir zar zor faaliyetlerini SUMOylation...

SUMO değişiklik başlangıçta protein istikrar1düzenleyen bir tersinir translasyonel modifikasyon (PTM) tespit edilmiştir. Doğrudan fiil ek olarak SUMO Ayrıca bir protein kovalent olmayan etkileşimi aracılığıyla SUMO etkileşim motifleri (SIMs)2tarafından eklenebilir. Src homoloji 2 (SH2) barındıran moleküller tarafından tyrosyl fosforilasyon bağlama veya phosphotyrosine bağlama benzer (PTB) etki alanları3,4, SUMO değişiklik için bir ek etkileşim platformu sağlar seçici İşe Alım SIM içeren efektör proteinler5,6. Ek olarak, düzenleme, sinyal iletimi, SUMOylation molekülleri remodeling Kromatin ve transkripsiyon faktörlerinin gen ifade7,8modüle için hizmet vermektedir. Genom-wide SUMOylation desen çalışmaları SUMO değişiklik olumlu9,10 ya da negatif10,11,12 transkripsiyonu ile ilişkili olduğunu ortaya koymuştur yönetmelik, SUMOylation paralogue özgüllüğü nedeniyle kısmen.
Orada üç büyük SUMO izoformlarının hediye memeli hücrelerinde conjugating protein için; Bu SUMO-1 ve son derece homolog SUMO-2 ve SUMO-3 (SUMO-2/3 adlandırılır)13içerir. SUMO genellikle hedef protein ψKxD/E uzlaşma motifi içinde lizin (K) kalıntı için Birleşik. SUMO E3 ligazlar SIM'de paralogue özgüllük14için sorumludur. E3 ligazlar yüzlerce içeren ubiquitination yolları aksine sadece birkaç SUMO E3 tespit ligazlar15olmuştur. SUMO E3 ligazlar (i) Ubc9 bağlama, bağlama (II) SUMO yan SIM etki alanı ve (III) Ubc9 SUMO transfer substrat üzerine geliştirmek amacıyla yeteneklerini de dahil olmak üzere özellikleri tanımlanır. E3 ligaz SUMO konjugasyon16için kesinlikle gerekli değildir, ancak substrat ve SUMO-paralogue özgüllük sağlar. Beri genellikle Toplam yüzey proteini küçük bir kısmını SUMO-modified, sadece SUMOylated proteinler vivo içinde tespiti her zaman bir meydan okuma olduğunu. Bu nedenle, doğru ve tekrarlanabilir deneyleri SUMO değişiklik hassas işlev aydınlatmak için gereklidir.
Vitro saf Ubc9 SUMOylation substrat proteinlerin katalizler yeteneğini değerlendirir, SUMOylation tahlil SUMO değişiklik17eğitim için iyi kabul edilen bir tahlil olduğunu. Ancak, çoğu durumda SUMO E3 ligaz aktivite tespit edilemez veya sadece mutasyona uğramış SUMO ligaz ile yüksek SUMO E3 ligaz aktivite ve düşük substrat özgüllüğü ile standart iletişim kuralı tarafından Ubc918 bol miktarda varlığı nedeniyle tespit edilebilir . Bu tahlil genel başarısı büyük ölçüde saf Ubc9 dikkatli titrasyon bağlıdır. Burada anlatılan vitro SUMOylation tahlil standart bir SUMOylation değiştirilir ( Tablo malzemelerigörmek) protokol. Kaposi sarkomu ilişkili herpesvirus (KSHV) yeniden etkinleştirme sırasında artan küresel SUMO değişikliği gözlenmesi bize SUMOylation up-yönetmelik için sorumlu olabilir viral SUMO E3 ligaz tanımlamak için istenir. Viral SUMO E3 ligaz K-bZIP etkileşen proteinler küçük ölçekli Gösterimin ardından, p53 roman bir substrat tespit edilmiştir. Bu protokol için ayrıntılı böcek hücreleri ifade ve vahşi-türü K-bZIP, viral bir SUMO E3 ligaz SUMO-2/3 özgüllük ile arınma adımları yer olarak gösteriyoruz. P53, iyi bilinen bir SUMO substrat SUMOylation geliştirmek için saflaştırılmış K bZIP yeteneği E1 ve E2 enzimler azaltılmış miktarda huzurunda değerlendirilir. Bu değiştirilmiş SUMOylation tahlil kullanarak, araştırmacılar güvenilir SUMO E3 ligaz yeteneklerini SUMOylate roman ya da bilinen yüzeylerde, hangi bir protein SUMOylation çalışmanın birincil adımdır tanımlayabilirsiniz. Ayrıca, bu yöntem düşük ligaz aktivite ama yüksek substrat özgüllüğü ile roman SUMO E3 ligazlar tanımlamaya yardım eder.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">pFastBac</td>
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			<td style="width:153px;">Invitrogen</td>
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			<td height="21" style="height:21px;width:363px;">competent cell&#160; E. coli DH5&#945;</td>
			<td style="width:153px;">Yeastern Biotech</td>
			<td style="width:119px;">FYE678-80VL</td>
			<td>competent E. coli cells A</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">E. coli DH10Bac</td>
			<td style="width:153px;">Invitrogen</td>
			<td style="width:119px;">10359-016</td>
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			<td height="21" style="height:21px;width:363px;">FugeneHD</td>
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			<td height="21" style="height:21px;width:363px;">T4 Ligase</td>
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			<td height="21" style="height:21px;width:363px;">CpoI (RsrII)</td>
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			<td height="21" style="height:21px;width:363px;">Bluo-gal</td>
			<td style="width:153px;">Thermo Scientific</td>
			<td style="width:119px;">B1690</td>
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			<td height="21" style="height:21px;width:363px;">IPTG</td>
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			<td height="21" style="height:21px;width:363px;">Grace&#8217;s Insect Medium</td>
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			<td style="width:119px;">11605094</td>
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			<td height="21" style="height:21px;width:363px;">Fetal Bovine Serum</td>
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			<td style="width:119px;">10082147</td>
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			<td height="21" style="height:21px;width:363px;">Gentamicin</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">15750060</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Ampicillin</td>
			<td style="width:153px;">Sigma-Aldrich</td>
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			<td height="21" style="height:21px;width:363px;">Kanamycin</td>
			<td style="width:153px;">Sigma-Aldrich</td>
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			<td height="21" style="height:21px;width:363px;">gentamicin</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">15710-064</td>
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			<td height="21" style="height:21px;width:363px;">tetracyclin</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">87128-25G</td>
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			<td height="21" style="height:21px;width:363px;">LB Broth</td>
			<td style="width:153px;">Merk</td>
			<td style="width:119px;">1.10285.0500</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">HEPES</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">H4034-100G</td>
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			<td height="21" style="height:21px;width:363px;">NaCl</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9888-5KG</td>
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			<td height="21" style="height:21px;width:363px;">KCl</td>
			<td style="width:153px;">Merk</td>
			<td style="width:119px;">1.04936.1000</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:363px;">Na2HPO4</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5136-500G</td>
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			<td height="25" style="height:25px;width:363px;">KH2PO4</td>
			<td style="width:153px;">J.T.Baker</td>
			<td style="width:119px;">3246-01</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">sodium dodecyl sulfate</td>
			<td style="width:153px;">Merk</td>
			<td style="width:119px;">1.13760.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">&#946;-mercaptoethanol</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">161-0710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">TRIS (Base)</td>
			<td style="width:153px;">J.T.Baker</td>
			<td style="width:119px;">4109-06</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:363px;">Non-fat milk</td>
			<td style="width:153px;">Fonterra</td>
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			<td height="21" style="height:21px;width:363px;">glycerol</td>
			<td style="width:153px;">J.T.Baker</td>
			<td style="width:119px;">2136-01</td>
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			<td height="21" style="height:21px;width:363px;">Triton X-100</td>
			<td style="width:153px;">Amresco</td>
			<td style="width:119px;">0694-1L</td>
			<td>detergent A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Tween 20</td>
			<td style="width:153px;">Amresco</td>
			<td style="width:119px;">0777-500ML</td>
			<td>detergent B</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Poloxamer 188 solution</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5556-100ML</td>
			<td>detergent C</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Protease Inhibitor Cocktail Tablet</td>
			<td style="width:153px;">Roche</td>
			<td style="width:119px;">04 693 132 001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">3x Flag peptide</td>
			<td style="width:153px;">Sigma</td>
			<td style="width:119px;">F4799</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">anti-FLAG m2 Magnetic beads</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8823</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">SUMOlink SUMO-1 Kit</td>
			<td style="width:153px;">Active Motif</td>
			<td style="width:119px;">40120</td>
			<td>standard SUMOylation protocol&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">SUMOlink SUMO-2/3 Kit</td>
			<td style="width:153px;">Active Motif</td>
			<td style="width:119px;">40220</td>
			<td>standard SUMOylation protocol&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">QIAquick Gel Extraction Kit</td>
			<td style="width:153px;">QIAGEN</td>
			<td style="width:119px;">28704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">QIAGEN Plasmid Mini Kit</td>
			<td style="width:153px;">QIAGEN</td>
			<td style="width:119px;">12123</td>
			<td>plasmid extraction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Polypropylene tubes</td>
			<td style="width:153px;">Falcon</td>
			<td style="width:119px;">352059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Petri Dish</td>
			<td style="width:153px;">Falcon</td>
			<td style="width:119px;">351029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Cell lifter</td>
			<td style="width:153px;">Corning</td>
			<td style="width:119px;">CNG3008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Loading tip</td>
			<td style="width:153px;">Sorenson BioScience</td>
			<td style="width:119px;">28480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">PVDF</td>
			<td style="width:153px;">PerkinElmer</td>
			<td style="width:119px;">NEF1002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Blotting filter paper</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">1703932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Mini slab gel apparatus (Bio-Rad Mini Protean II Cell)</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">1658001 EDU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">1703940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Pierce ECL Western Blotting</td>
			<td style="width:153px;">Thermo</td>
			<td style="width:119px;">32106</td>
			<td>ECL reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:363px;">suspension mixer</td>
			<td style="width:153px;">Digisystem laboratory instruments&#160; Inc.</td>
			<td style="width:119px;">SM-3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">orbital shaker</td>
			<td>Kansin instruments Co.</td>
			<td>OS701</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:363px;">ImageQuant LAS 4000 
			biomolecular imager</td>
			<td style="width:153px;">GE Healthcare</td>
			<td style="width:119px;">28955810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Sf9</td>
			<td style="width:153px;">Thermo Scientific</td>
			<td>B82501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">anti-p53 antibody</td>
			<td>Cell Signaling</td>
			<td>#9282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">anti-rabbit antibody</td>
			<td>GE Healthcare</td>
			<td>NA934-1ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro sumoylation assay to study sumo e3 ligase activity

Küçük Ubikuitin benzeri değiştirici (SUMO) değişiklik öncelikle protein-protein etkileşimleri oransal aracılığıyla sinyal iletimi aracılık eder önemli bir translasyonel modifikasyon (PTM) olduğunu. Ubikuitin değişikliklere benzer, SUMOylation enzim (SAE1/SAE2) E1 etkinleştirme, E2-konjugasyon enzim (Ubc9) ve E3-ligaz (Yani, PIA aile, RanBP2 ve Pc2) gibi bir sıralı enzim çağlayan tarafından yönlendirilir. Ama tepki değil ancak, ubiquitination, bir E3 ligaz gerekli olmayan farklıdır SUMO konjugasyon için hassas ve etkinliği sağlamak. SUMOylation tarafından modifiye proteinleri vivo içinde tahlil immunoprecipitation üzerinden substrat özel antikorlar ve immunoblotting SUMO özel antikorlar ile belirlenebilir. Ancak, protein SUMO E3 ligaz faaliyet gösteri arıtılmış enzim, substrat ve SUMO proteinler kullanarak SUMOylation deneyleri in vitro sulandırma gerektirir. Vitro reaksiyonlar genellikle SAE1/SAE2 ve Ubc9, yalnız SUMO konjugasyon için yeterli olduğundan, SUMOylation geliştirme sözde E3 ligaz tarafından her zaman tespit etmek kolay değildir. Burada, bir değiştirilmiş vitro sürekli olarak yeniden düzenlendi vitro sistemini kullanarak SUMO değişiklik tanımlar SUMOylation Protokolü açıklar. Catalytically etkin K-bZIP, viral bir SUMO-2/3 E3 ligaz arındırmak için adım adım bir protokol de sunulmaktadır. Arıtılmış K bZIP SUMOylation faaliyetlerinin p53, SUMO tanınmış bir hedef gösterilir. Bu protokolü yalnızca roman SUMO E3 ligazlar elucidating için aynı zamanda SUMO paralog olmalarından ifşa için istihdam edilebilir değil.

Üretici tarafından sağlanan bilgilere göre E1 ve E2 enzim SUMOylation tahlil standart miktarı 50 şey nM ve 500 nM, anılan sıraya göre. E2 en az miktarda enzim SUMOylate p53 yapabiliyor Ubc9 conjugating ilk bir vitro SUMOylation tahlil tarafından tespit edilmiştir. Beşte biri Ubc9 standart vitro SUMOylation tahlil protokolü kullanılan miktarı verimli bir şekilde SUMOylate p53 için mümkün olduğu kadar düşük (şekil 1<str...

Watch this Scientific Journal Video about In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity at JoVE.com

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Ubikuitin ligazlar, birkaç E3 SUMO ligazlar tespit edilmiştir. Bu değiştirilmiş vitro SUMOylation Protokolü roman SUMO E3 ligazlar bir vitro sulandırma tahlil tarafından tespit edebilmek.

Vitro SUMOylation SUMO E3 ligaz etkinlik eğitim için tahlil

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...

in-vitro-sumoylation-assay-to-study-sumo-e3-ligase-activity

Research

JoVE Journal

Biology

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

9.3K Görüntüleme.  National Yang-Ming University. Ubikuitin ligazlar, birkaç E3 SUMO ligazlar tespit edilmiştir. Bu değiştirilmiş vitro SUMOylation Protokolü roman SUMO E3 ligazlar bir vitro sulandırma tahlil tarafından tespit edebilmek.

Video: In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

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

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

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

Kertenkeleler Hareket Hız ve Yön Visual Hassasiyet Test

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

Biyoloji

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

Bir In vitro FluoroBlok Tümör Invasion Testi

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

LRRK2 Kinaz Aktivitesi Assaying In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

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

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

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

KRİYOPREZERVE Numune Kurtarma için Do-It-Yourself Aygıt yanlışlıkla Kriyojenik Depolama Tankları içine Dropped

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

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Endotel Hücre için Tüp Deneyi birleşimi<em&gt; İn Vitro</em&gt; Anjiyogenezin Çalışması

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro Metilasyona Tahlil Protein Arginin metilasyon Okuyacak

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

Vitro Tahlil Fosfatidiletanolamin Metiltransferaz Aktivite Tedbir

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

ölçme<em&gt; İn Vitro</emEnzimatik Karakterizasyonu için&gt; ATPaz Aktivitesi

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

Vivo Algılama ve Rb Protein SUMOylation insan hücrelerinde analizi

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

Genom kapsamındaki bir tanımlama ve ifade Meta-Analizi ATL E3 Ubikuitin ligaz gen ailesinin asma için kapsamlı iş akışı

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...