Danilo Maddalo bir araştırma bursu, Karlsruhe Teknoloji Enstitüsü Genç Araştırmacı Grubu (YIG) olarak bir alıcı.

1. Hücreleri Seeding <ol><li> Başlamadan önce, 37 az bir su banyosu içinde, kültür ortamı, PBS 1X ve tripsin ısınmak ˚ C </li><li> Kuluçka hücrelerini alın ve orta aspire. </li><li> 5 ml PBS 1X nazikçe yıkayın hücreleri geçerlidir. </li><li> PBS 1X aspire tripsin EDTA% 0,025 içeren 1 ml uygulayın. </li><li> Tripsin eşit bir dağılıma sahip olduğu plaka hafifçe döndürün. </li><li> 37 inkübatör hücrelerin yerleştirin ˚ C (hücre tipine bağlı olarak 5-10 dakika). Zaman zaman kontrol hücreleri plaka sallayarak ayrılmış. </li><li> 10-20 ml orta hücrelerin 50 ml şahin tüp içinde süspanse edin ve onları toplamak. </li><li> Beckman sayacı kullanarak hücre sayımı. Hücrelerin sayısı yaklaşık% 60-70 oranında bir izdiham sağlamak için yeterli olmalıdır. </li><li> Plate hücreleri ve 6-8 saat süreyle tohum onlara izin verir. </li></ol> 2. TransInfeksiyon <ol><li> Transfeksiyon için önce 37 ˚ C&#39;ye bir su banyosu içinde bir kısım serum orta ısınmak </li><li> X-treme Gen 9 oda sıcaklığına getirin. </li><li> 2 Eppendorf tüpler serum serbest orta (1 tüp = 1 transfeksiyon) pipetleyin. </li><li> Pipetleyin ucu, 5 dakika, Eppendorf ve inkübe plastik duvar temas edip etmediğini, serum serbest orta ödeyerek dikkat X-treme Gen 9. Bu arada iki DNA karışımlarını hazırlamak: Bir tüp içinde, başka bir tüp renilla, ateş böceği ve moleküler şaperon renilla, ateş böceği ve boş bir vektör kontrolü için plazmid karıştırın. </li><li> + X-treme Gen 9, vortex ve 20 dakika oda sıcaklığında inkübe içeren serum serbest ortam her Eppendorf DNA karışımını ekleyin. </li><li> 37, 24 saat boyunca her plaka ve inkübe üzerine damla damla her karışımı ° C ve% 5 CO 2 </li></ol> 3. Yarma hücreleri <ol><li> 24 saat sonra transfeksiyon, tryps1.4 &#39;de açıklandığı gibi hücrelere inize - 1.6 ve% 60-70 oranında son bir confluency 6 plaka onları sulandırmak. </li><li> Olmayan şok kontrol için bir tabak hazırlayın ve analiz edilmesi gereken kurtarma zamanı noktaları gibi birçok tabak, Şekil gösterdi. 2. </li></ol> 4. Isı şoku <ol><li> Isı çarpması için, önceden ılık serum içeren bir su banyosunda 37 ° orta ˚ C </li><li> 20 mM nihai konsantrasyonu 20 mg / ml ve MOPS serum içeren orta Siklohekzimidsiz içinde seyreltilmesi hazırlayın &#39;ısı şok tampon. </li><li> Şimdi inkübatör tabak ve orta aspire. </li><li> &#39;Isı şok tampon&#39; her iyi 1 ml ekleyin ve 37 az 30 dakika boyunca inkübe ˚ C ve% 5 CO 2 (aynı zamanda bu tedavi referans plaka). Bu, de novo protein sentezi durur. </li><li> Bu arada bir su banyosu anahtarı ve temperature ˚ C 45 </li><li> Inkübasyondan sonra, daha önce şeritler halinde kesilmiş plakalar Parafilm kapak mühür. Referans plaka (refolfed ateşböceği% 100) inkübatör bırakılır. </li><li> 45 ˚ C &#39;de 30 dakika boyunca plakalar inkübe edin. </li><li> Su banyosu plakaları çıkarın parafilm kaldırmak ve kurtarma izin inkübatör geri tabak koyun. </li><li> Her zaman noktası hasat hücrelerin az ve 4 için 2000Xg ˚ C&#39;de 1 dakika santrifüj PBS 1X yıkayın </li></ol> 5. Hücre parçalama ve lusiferaz tahlil <ol><li> Verimli bir hücre erimesi için, sıvı nitrojen içinde hücre pelletini dondurmak oturtun. </li><li> Bir cam tüp çift distile su 1:05 seyreltilmiş pasif lizis tamponu 200 ul ekleyin. </li><li> 96 plaka 30 ul hücre lizat ekleyin. Her örnek, üç nüsha olarak pipetlenir. </li><li> Sadece olmayan şok &#39;referans örnek olacakBu adım, eşit transfeksiyon etkinliğini kontrol etmek için kullanılan bu yana, renilla lusiferaz aktivitesini okuyun. </li><li> Test için çözümler hazırlayın. Luciferase çözümü için Gly-Gly tampon son konsantrasyonu 0.2 mM luciferase sulandırmak. Reaksiyon tamponu için Gly-Gly tampon 1mm son konsantrasyonu DTT ve ATP sulandırmak. Coelenterazine reaksiyon tamponu için, nihai konsantrasyonu 0.2 mcM coelenterazine tampon coelenterazine sulandırmak. Luciferase çözüm ve coelenterazine reaksiyon tamponu ışık korumalı tutun. </li><li> Şimdi luminometreye 96-plaka yeri ve programı &#39;Wallac 1420 İş İstasyonu&#39; başlatmak. </li><li> Coelenterazine çözeltisi içeren şahin tüpe Pump1 tanıtın. Hiç ışık tüpü ile temas böylece kapağını kapatın. </li><li> Seçeneği &#39;Dispenser bakım menüden seçiniz ve Pump1 işaretleyiniz. </li><li> &#39;Doldur&#39; seçeneğini seçin. </li><li> Menüde &#39;plaka düzeni seçeneği düzenlemek için protokol ve sonuç keşfedin. Üzerinde protokol ve çift tıklayarak seçin. Şimdi kuyu okunacak seçin. </li><li> &#39;Wallac Müdürü&#39; geri dönün ve &#39;Başlat&#39; düğmesine tıklayın. Renilla lusiferaz ve substrat coelenterazine arasındaki reaksiyon pik, 482 nm dalga boylu ışık yayar. </li><li> Okuduktan sonra, &#39;Dispenser bakım gidin ve geri kalan tüm çözüm tüp serbest bırakmak için&#39; Boş &#39;ve Pump1 seçin. </li><li> Şimdi, su içeren bir şahin tüp tüp taşımak ve &#39;Flush&#39; işaretleyiniz. </li><li> Kızarma adımdan sonra, &#39;Boş&#39; seçerek tüp boş. </li><li> Reaksiyon tampon şahin tüp Pump1 ve luciferase şahin tüp Pump2 biri tüp yerleştirin. </li><li> Plaka düzenini değiştirin. </li><li> &#39;Di Pump1 ve Pump2&#39; Doldur &#39;seçinSpenser Bakım &#39;menüsünden. </li><li> Protokolü seçin ve okumaya başlayın. Firefly lusiferaz ve substrat luciferase arasındaki reaksiyon, 560 nm arasında bir zirve dalga boyuna sahip bir ışık yayar. </li><li> Okuma sonunda Pump1 ve Pump2 Flush-Boş Boş işlemi tekrarlayın. </li><li> Sonuçlar &#39;Wallac 1420 Explorer&#39; artık mevcuttur. </li><li> Her deneyde bir test numarası ile ilişkili, başlangıç ​​ve bitiş tarihi kullanıcı adınızı. </li><li> Sonuçlar artık bir Excel dosyası olarak ihraç edilebilir. </li><li> 1: hesaplamalar için, hücreleri üç gruba ayrılır. olmayan şok referans örnekleri (% 100 ateşböceği lusiferaz refolded set); 2. şok moleküler hastabakıcı ve ısı olmadan hücreler; 3. hücrelerin moleküler şaperon ısı ve şok. </li></ol> 6. Temsilcisi Sonuçlar Protein refolding sistem yanlısı çalışıp çalışmadığını test etmek için bu nedenle iyileşme süresi doğrudan ilgili<html> Perly bir tahlil, farklı zaman noktalarında hücrelerin toplanması yapılacak. Doğrudan bir korelasyon zaman / refolding yüzdesi, deney düzgün bir şekilde gerçekleştirilir ve bu nedenle bir sınırlayıcı faktör temsil olduğunu gösterir. Ancak her zaman ısı şokundan sonra refolding doyurarak bir olaydır ve bu nedenle uygun bir zaman ders analizi, herhangi bir deney başlamadan önce yapılmalıdır dikkate alınmalıdır. <img src="/files/ftp_upload/3540/3540fig1.jpg" alt="Şekil 1"/> Şekil 1. Deney Bakış. In vivo refolding tayininde tamamlanması 3 ila 4 gün gerektirir. Gün 1 hücreleri numaralı seribaşı ve transfekte. Ertesi gün, hücreleri daha küçük bir biçime parçalı ve 3. günde ısı şok. Bu noktada sağ hücreler hasat sonrası lusiferaz tahlil gerçekleştirmek için veya -80 ° C&#39;de hücre pelletini kaydetmek ve bir an tahlil gerçekleştirmek mümkün _content &quot;&gt; <img src="/files/ftp_upload/3540/3540fig2.jpg" alt="Şekil 2"/> Şekil 2. Hücre bölünmesi. Gününde 2 hücreleri 6 plaka ayrılır. Her transfeksiyon aynı plaka üzerindeki tüm transfeksiyon sahip olmak için iyi bir seyreltilmiş olacaktır. Her plaka belirli bir iyileşme süresi artı olmayan bir şok referans plaka karşılık gelir. <img src="/files/ftp_upload/3540/3540fig3.jpg" alt="Şekil 3"/> Şekil 3. Temsilcisi iyi bir sonuç. Refolding, A. Yüzde doğrudan ateşböceği lusiferaz aktivite ile ölçülür. Her çubuk grafik, farklı kurtarma zaman noktalarında moleküler şaperon varlığı yokluğu (siyah çubuklar) ve (kırmızı bar) refolding yüzdesini temsil eder. B. yokluğu (siyah çizgi) ve hastabakıcı varlığı (kırmızı çizgi) ve refolding arasında korelasyon. R kare değerleri arasında iyi bir korelasyon göstermektedir. Fig.3A hücrelerin yokluğunda ısı şok (siyah çubuklar) 15, 30, 45, 60 ve 120 dakika sonra ve moleküler şaperon varlığı (kırmızı bar) toplandı bir temsilci sonuçları gösterilir. Yüzde refolded, uzamış iyileşme süresi ve moleküler şaperon transfeksiyon lusiferaz artar. Refolding ve zaman kontrolü (Şekil 3B, siyah çizgi) ve moleküler hastabakıcı-transfekte hücreler (Şekil 3B, kırmızı çizgi) arasındaki ilişki gösterilmiştir. <img src="/files/ftp_upload/3540/3540fig4.jpg" alt="Şekil 4"/> Şekil 4 Temsilcisi kötü bir sonuç. A. refolding, Yüzde doğrudan luciferase aktivitesi ölçülür. Her çubuk grafik, farklı kurtarma zaman noktalarında moleküler şaperon varlığı yokluğu (siyah çubuklar) ve (kırmızı bar) refolding yüzdesini temsil eder. Yokluğunda ve varlığında zaman ve refolding arasında B. Korelasyon (kırmızı lhastabakıcı ine). R kare değerleri kötü bir korelasyon göstermektedir. Şekil 4A düzgün yapılan bir deneyin temsili bir sonuç göstermektedir. Kontrol (siyah çubuklar) ve moleküler hastabakıcı-transfekte hücreleri (kırmızı bar) her ikisi de zaman refolding protein bir artış göstermemiştir. Buna ek olarak, moleküler şaperon transfeksiyon refolding bir artışa yol açmamıştır. Bu zayıf bir korelasyon Şekil teyit edilir. Kontrolü (siyah çizgi) ve moleküler hastabakıcı-transfekte hücreler (kırmızı çizgi) için sırasıyla 0,6619 ve 0,1882 r kare değeri gösteren 4B.

<ol>
	<li>Bukau, B., Weissman, J., Horwich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperones+and+protein+quality+control.">Molecular chaperones and protein quality control.</a> Cell. 125, 443-443 (2006).</li><li>Ritossa, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+the+heat+shock+response.">Discovery of the heat shock response.</a> Cell Stress Chaperones. 1, 97-97 (1996).</li><li>Hendrick, J. P., Hartl, F. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperone+functions+of+heat-shock+proteins.">Molecular chaperone functions of heat-shock proteins.</a> Annu. Rev. Biochem. 62, 349-349 (1993).</li><li>Fuqua, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+proteins+and+drug+resistance.">Heat shock proteins and drug resistance.</a> Breast. Cancer. Res. Treat. 32, 67-67 (1994).</li><li>Guzhova, I., Margulis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp70+chaperone+as+a+survival+factor+in+cell+pathology.">Hsp70 chaperone as a survival factor in cell pathology.</a> Int. Rev. Cytol. 254, 101-101 (2006).</li><li>Whitesell, L., Lindquist, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP90+and+the+chaperoning+of+cancer.">HSP90 and the chaperoning of cancer.</a> Nat. Rev. Cancer. 5, 761-761 (2005).</li><li>Konstantinopoulos, P. A., Papavassiliou, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=17-AAG:+mechanisms+of+antitumour+activity.">17-AAG: mechanisms of antitumour activity.</a> Expert. Opin. Investig. Drugs. 14, 1471-1471 (2005).</li><li>Galluzzi, L., Giordanetto, F., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+HSP70+for+cancer+therapy.">Targeting HSP70 for cancer therapy.</a> Mol. Cell. 36, 176-176 (2009).</li><li>Ozacmak, V. H., Barut, F., Ozacmak, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melatonin+provides+neuroprotection+by+reducing+oxidative+stress+and+HSP70+expression+during+chronic+cerebral+hypoperfusion+in+ovariectomized+rats.">Melatonin provides neuroprotection by reducing oxidative stress and HSP70 expression during chronic cerebral hypoperfusion in ovariectomized rats.</a> J. Pineal. Res. 47, 156-156 (2009).</li><li>Schroder, H., Langer, T., Hartl, F. U., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DnaK,+DnaJ+and+GrpE+form+a+cellular+chaperone+machinery+capable+of+repairing+heat-induced+protein+damage.">DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.</a> EMBO. J. 12, 4137-41 (1993).</li><li>Thulasiraman, V., Matts, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+geldanamycin+on+the+kinetics+of+chaperone-mediated+renaturation+of+firefly+luciferase+in+rabbit+reticulocyte+lysate.">Effect of geldanamycin on the kinetics of chaperone-mediated renaturation of firefly luciferase in rabbit reticulocyte lysate.</a> Biochemistry. 35, 13443-13443 (1996).</li><li>Howarth, 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=Hsp40+molecules+that+target+to+the+ubiquitin-proteasome+system+decrease+inclusion+formation+in+models+of+polyglutamine+disease.">Hsp40 molecules that target to the ubiquitin-proteasome system decrease inclusion formation in models of polyglutamine disease.</a> Mol. Ther. 15, 1110-1110 (2007).</li><li>Nollen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bag1+functions+in+vivo+as+a+negative+regulator+of+Hsp70+chaperone+activity.">Bag1 functions in vivo as a negative regulator of Hsp70 chaperone activity.</a> Mol. Cell. Biol. 20, 1083-1083 (2000).</li></ol>

Yazarlar ifşa etmek başka bir şey var.

Bu çalışmada, moleküler şaperonlar içi refolding aktivitesini ölçmek için bir protokol sunulmuştur. Şekil bakış gösterildiği gibi tüm testi 3 ila 4 gün içinde yapılabilir. 1. Ateş böceği ve renilla lusiferaz tarafından üretilen ışık sinyali sağlamlık ve doğrusallık, tekrarlanabilirlik protokol için sağlam bir temel temsil eder. Testin kritik adım, moleküler hastabakıcı ve ateşböceği lusifer...

Bu deneme için bir Luminometre formu Perkin Elmer &#39;1420 Lüminesans Sayaç &#39;Vektör Light&#39; kullanıldı Luminometre Programları: Renilla: Pompa 1, 100 ul enjeksiyon Reaksiyon Tamponu: Pump1, 70 ul enjeksiyon Luciferase: Pump2, 30μl enjeksiyon Tamponlar çift distile su bu stok solüsyonları hazırlamak için: <ul style="list-style:none"><li> 1M MgSO 4: 1 litre 246,48 g MgSO 4 * 7H 2 O </li><li> - 0,5 M EGTA: 1 litre 190,18 g EGTA </li><li> - 1M KH 2 PO 4: 1 litre 136,09 g KH 2 PO 4 </li><li> - 1M K2 HPO 4: 228,23 g K 2 HPO 4 * 1 litre 3H 2 O </li><li> - 5M NaCl: 1 litre 292,2 g NaCl </li><li> - 0,5 M EDTA: 1 litre 186,12 g EDTA * 2H 2 O &nbsp;</li></ul> Gly-Gly-tampon: <ul style="list-style:none"><li> - 25mm Glycylglycin </li><li> - 15mm MgSO 4 </li><li> - 4mm EGTA </li></ul> Için 1 litre Glycylglycin kullanımı 3,3 g, 1M MgSO4 çözüm ve 0,5 M EGTA çözüm, 8 ml 15ml. Su içinde çözün, 7,8 pH ayarlamak ve toplam hacim 1 litre getirmek. Renilla / Coelenterazine tampon reaksiyon tamponu: <ul style="list-style:none"><li> &amp; Nbsp; 13,4 mM KH 2 PO 4 </li><li> KH 2 PO 4 - 86,6 mM </li><li> - 0,5 M NaCl </li><li> - 1mm EDTA </li></ul> 1 litre 1M KH 2 PO 4 çözümü kullanmak 13,4 ml, 86,6 ml, 5M NaCl çözeltisi ve 2 ml 0,5 M EDTA çözeltisi 100ml 1M bir K 2 HPO 4 çözüm . <table border="1"><tr><td></td><td> Şirket </td><td> Katalog / Ürün Numarası: </td></tr><tr><td> DMEM (Dulbecco&#39;nun Modifiye Eagle Orta 1X) </td><td> Gibco </td><td> 41966029 </td></tr><tr><td> Fetal Bovine Serum Altın </td><td> PAA </td><td> A15-151 </td></tr><tr><td> X-treme Gen 9 DNA Transfeksiyon Reaktif </td><td> Roche </td><td&gt; 06365809001 </td></tr><tr><td> PBS Dulbecco&#39;nun Fosfat Tuzlu 1X Tamponlu </td><td> Gibco </td><td> 14190-094 </td></tr><tr><td> MOPS 3 - (N-morfolino) propan sülfonik asit </td><td> Sigma-Aldrich </td><td> M1254 </td></tr><tr><td> Cycloheximide </td><td> Sigma-Aldrich </td><td> C7698 </td></tr><tr><td> Pasif Lizis Tampon 5X </td><td> Promega </td><td> E194A </td></tr><tr><td> Glycylglycin EGTA MgSO 4 (MgSO 4 * 2 O 7H) </td><td> Sigma-Aldrich Roth Roth </td><td> G7278 3054,2 P027.1 </td></tr><tr><td> KH 2 PO 4 K 2 HPO 4 (K 2 HPO 4 * 3H 2 O) NaCl EDTA </td><td> Roth Roth Roth Roth </td><td> 3904,1 6878,2 3957,1 8043,1 </td></tr><tr><td> Luciferase Firefly </td><td> Biosynth </td><td> L8200 </td></tr><tr><td> Coelenterazine </td><td> Biosynth </td><td> C7000 </td></tr><tr><td> DTT (dithiothreitol) </td><td> Roth </td><td> 6908,2 </td></tr><tr><td> ATP (adenozin trifosfat) </td><td> Roche </td><td> 10519987001 </td></tr></table>

intracellular refolding assay

Bu protokol, bir hücre tabanlı bir sistem ve inhibitör / uyarıcı aktiviteye sahip bileşikler olası etkileri içinde moleküler şaperonlar enzimatik aktivitesini ölçmek için bir yöntem açıklanır. Moleküler şaperonlar 1 katlama protein yönetmelikte yer alan proteinler ve ısı şok 2, besin açlık ve kimyasallar / zehirler 3 maruz kalma gibi stres hakaretler üzerine hücre sağkalım teşvik çok önemli bir rol var. Bu nedenle şaperonlar tümör gelişimi, kanser hücreleri 4 chemioresistance yanı sıra nörodejenerasyon 5 gibi etkinliklere yer bulunur. Bu nedenle, bu enzimlerin aktivitesini inhibe veya teşvik etmek için küçük moleküllerin tasarımı, kanser tedavisi 7 ve nörodejeneratif hastalıkların 9 için en çok çalışılan stratejileri biridir. Burada açıklanan belirli bir moleküler şaperon refolding aktivitesini ölçmek ve comp etkisini araştırmak için tahlil imkanı sunarfaaliyet ounds. Bu yöntemde, incelenen moleküler şaperon gen ateşböceği lusiferaz gen ekspresyon vektörü kodlama ile birlikte transfekte. Zaten denatüre ateşböceği lusiferaz moleküler şaperonlar 10,11 refolded olabileceği tarif edilmiştir. Transfeksiyon kontrolü normalleştirilmesi gibi, renilla lusiferaz gen için bir vektör kodlama transfekte. Bu protokolü açıklanan tüm transfections HEK-293 hücrelerinde X-treme Gen 11 (Roche) ile yapılmaktadır. İlk adımda, protein sentezi, Siklohekzimidsiz hücrelerin tedavi inhibe edilir. Bundan sonra açığa çıkmasını protein 30 dakika boyunca 45 ° C&#39;de ısı şok indüklenir. 37 ° kurtarma üzerine ° C, proteinler yeniden katlanmış ve onların aktif konformasyon ateşböceği lusiferaz faaliyet okuma olarak kullanılır: daha fazla ışık olacak, daha fazla protein orijinal konformasyon yeniden kazanmış olacaktır. Isıl şok hücreleri referans (refolded,% 100 olarak belirlenmiştirlusiferaz).

Watch this Scientific Journal Video about Intracellular Refolding Assay at JoVE.com

Intracellular Refolding Assay

Bu protokol ısı şok sonra hücre içi protein refolding ölçmek için bir yöntem açıklanmıştır. Bu yöntem, faaliyetlerini etkilemek için moleküler şaperonlar ve bunların eş-faktörleri veya bileşikleri gibi foldases incelemek için kullanılabilir. Firefly lusiferaz aktivitesini muhabir olarak hastabakıcı refolding aktivitesini ölçmek için kullanılır.

Hücre içi Refolding Testi

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

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JoVE Journal

Biology

14.1K Görüntüleme.  Karlsruhe Institute of Technology. Bu protokol ısı şok sonra hücre içi protein refolding ölçmek için bir yöntem açıklanmıştır. Bu yöntem, faaliyetlerini etkilemek için moleküler şaperonlar ve bunların eş-faktörleri veya bileşikleri gibi foldases incelemek için kullanılabilir. Firefly lusiferaz aktivitesini muhabir olarak hastabakıcı refolding aktivitesini ölçmek için kullanılır.

Video: Intracellular Refolding Assay

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Astrositler Plazma Membran ve Küresel Hücre içi kalsiyum Dynamics civarında Ölçüm

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Biyoloji

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Fare Pankreas Adacık İzolasyonu ve hücre içi cAMP Belirlenmesi için Bir Yöntem

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

İzlenmesi protein refolding için birleştiğinde Tahliller<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Floresan Proteinler ifade Canlı Farelerde Görüntüleme hücrealtı Yapıların Intravital Mikroskopi

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by &#946; and &#947;-secretases. Despite the importance of A&#946; to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.
While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the &#947;-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.
We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.

While the transport of cell surface proteins is relatively easily studied, visualizing the trafficking of intracellular proteins is much more difficult. Here, we use constructs incorporating photoactivatable GFP and demonstrate a method to accurately follow the amyloid precursor protein from the Golgi apparatus to down-stream compartments and follow its clearance.

Photoactivatable GFP ile APP Hücre içi Ticareti Görüntüleme

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the ...

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, ...

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni2+-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

Saflaştırma ve Amiloid fibrillerin kadar yeniden katlama (His)<sub&gt; 6</sub&gt; etiketli Rekombinant Shadoo Protein Eklenmesi Organları olarak ifade<em&gt; E.coli</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

Hücre içi Komşu maruz kalan hücreler, apoptotik hücre ölümü ile etkileşim yoluyla canlı hücreler İndüklenen sinyal olaylarının tanımlanması

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Drosophila melanogaster Malpighi tübül epiteli optik Quantification intrasellüler pH floresan genetik olarak kodlanmış pH göstergesi

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

Dinamik için objektif-ücretsiz Video mikroskobu ve kantitatif analiz yapisan hücre kültürü

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...