Name: monitoring of ubiquitin-proteasome activity in living cells using a degron (dgn)-destabilized green fluorescent protein (gfp)-based reporter protein
Uploaded: 2012-11-10T14:00:00
Description: Watch this Scientific Journal Video about Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein at JoVE.com

Bu çalışma tarafından finanse edildi: Avusturya Bilim Vakfı (FWF), Avrupa Komisyonu Entegre Projeler MiMAGE ve PROTEOMAGE, Hollanda Girişimi Genomics tarafından Yaşlanma (NFN S93) Ulusal Araştırma Ağı / Bilimsel Araştırma Hollanda Teşkilatı (NGI / NWO, 05.040.202 ve 050 - 060-810 NCHA), AB Mükemmellik Ömrü (FP6 036.894) Network finanse ve Genomik üzerinde Yenilik Odaklı Araştırma Programı (SenterNovem; IGE01014 ve IGE5007).

1. Plazmid İnşaat <ol><li> Sipariş özel oligo-nükleotidler DGN (ACKNWFSSLSHFVIHL 11) ve dgnFS (HARTGSLACPTSSSICE) için kodlama ve DGN / dgnFS (Şekil 1) ile GFP füzyon elde etmek için pEGFP-C1 vektör içine Arter. </li><li> Pentr Yönlü TOPO Klonlama Kit protokol ve pLenti6/V5 Yönlü TOPO Klonlama Kiti (Şekil 6) ile devam göre PCR ile GFP-DGN ve GFP-dgnFS için kodlama sırasını yükseltin. </li></ol> 2. Virüs Üretimi <ol><li> Onla...

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Ubikuitin-proteazom aktivitesi için bir muhabir substrat olarak yeşil floresan protein (GFP) kullanarak ilk yayın 2000 12 yayımlanmıştır. O zamandan beri, GFP hücresel faaliyetler, özellikle ubikuitin-proteazom işlemini görselleştirmek için ortak bir araç haline gelmiştir. In vivo ubikuitin-proteazom etkinliğini izlemek için bir GFP tabanlı muhabiri ile bir transgenik fare modelinde 13 girmiştir. In vivo araştırmalar bu yayın ile ilgili ek 14</s...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> pEGFP-C1 vektör </td><td> BD Bioscience Clontech </td><td> 6084-1 </td></tr><tr><td> pentr Yönlü TOPO Klonlama Kiti </td><td> Invitrogen </td><td> K2400-20 </td></tr><tr><td> pLenti6/V5 Yönlü TOPO Klonlama Kiti </td><td> Invitrogen </td><td> V496-10 </td></tr><tr><td> Lipofektamin 2000 Reaktif </td><td> Invitrogen </td><td> 11668019 </td></tr><tr><td> DMEM </td><td> Sigma </td><td> D5546 </td></tr><tr><td> PVDF filtre (Rotilabo-Spritzenfilter) </td><td> Roth </td><td> P667.1 </td></tr><tr><td> Polietilen glikol </td><td> Sigma </td><td> P2139 </td></tr><tr><td> NaCl </td><td> Merck </td><td> 1.06404.1000 </td></tr><tr><td> Dulbecco Fosfat Sali Tamponlune 1x (PBS) </td><td> Invitrogen </td><td> 14190 </td></tr><tr><td> hexadimethrine bromür </td><td> Sigma </td><td> 10,768-9 </td></tr><tr><td> Blasticidin </td><td> Invitrogen </td><td> R21001 </td></tr><tr><td> Kristal viyole </td><td> Sigma </td><td> C3886 </td></tr><tr><td> FACS tüpler </td><td> BD Biosciences </td><td></td></tr><tr><td> Penisilin Streptomisin (Kalem-Strep) </td><td> Invitrogen </td><td> 15140130 </td></tr><tr><td> L-glutamin 200 mM </td><td> Invitrogen </td><td> 25030024 </td></tr><tr><td> Sığır fetüs serumu (FBS) </td><td> Biochrom AG </td><td> S0115 </td></tr><tr><td> MEM Temel Olmayan Amino Asitler (NEAA) 100x </td><td> Invitrogen </td><td> 11140035 </td></tr><tr><td> MEM 100 mM sodyum piruvat </td><td> Invitrogen </td><td> 11360039 </td></tr><tr><td> D-(+)-glikoz (% 45) </td><td> Sigma </td><td> G8769 </td></tr><tr><td> Genetisin </td><td> Invitrogen </td><td> 11811023 </td></tr><tr><td> CaCl2 </td><td> Merck </td><td> C5080 </td></tr><tr><td> Hepes </td><td> Sigma </td><td> H3375 </td></tr><tr><td> Tripsin-EDTA (% 0.05) </td><td> Invitrogen </td><td> 25300054 </td></tr></tbody></table>

monitoring of ubiquitin-proteasome activity in living cells using a degron (dgn)-destabilized green fluorescent protein (gfp)-based reporter protein

Proteazom anormal, katlanan hasar görmüş ya da oksitlenmiş proteinler 1, 2, proteolitik bozulması ile ilgili ana hücre içi organel olup. Proteazom faaliyet Bakım hücrenin stres yanıtı 3, hücre döngüsü kontrolü ve hücresel farklılaşma 4 veya bağışıklık sistemi yanıtı 5, gibi birçok temel hücresel süreçler karışmıştı. Ubiquitin-proteasome sistemi fonksiyon bozukluğu tümörler ve nörodejeneratif hastalıklar, 4, 6 ve gelişimi ile ilgili olmuştur. Buna ek olarak, proteazom aktivitesinde bir azalma hücresel yaşlanma ve organizma yaşlanma 7, 8, 9, 10 bir özellik olarak tespit edildi. Burada, bir GFP-DGN füzyon proteini kullanarak canlı hücrelerde ubikuitin-proteazom aktivitesini ölçmek için bir yöntem mevcut. Birincil hücreler canlı ubikuitin-proteazom etkinliğini izlemek edebilmek için, tamamlayıcı DNA (yeşil floresan protein (GFP)-DGN füzyon proteini kodlayan inşa GFP-DGN, kararsız) ve bir çerçeve kayması mutasyon (GFP-dgnFS, kararlı 11) taşıyan bir varyant lentiviral ekspresyon vektörleri içine yerleştirilir. Bu hücre tipi ya da donör yaşı bağımsız çok yüksek transfeksiyon verimliliği garanti çünkü geleneksel transfeksiyon teknikleri üzerinde bu tekniği tercih. GFP-dgnFS (kararlı) protein ve protazom inhibitörünün yokluğunda veya varlığında istikrarsız protein (GFP-DGN) tarafından gösterilen floresan arasındaki fark, her bir hücre soyu içinde ubiquitin-proteasome aktivitesi tahmin etmek için de kullanılabilir. Bu farklılıklar Epifloresans mikroskopi ile takip edilebilir ya da akış sitometrisi ile ölçülebilir.

Watch this Scientific Journal Video about Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein at JoVE.com

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

Canlı hücreler içinde ubiquitin-proteasome etkinliğini izlemek için bir yöntem tarif edilmektedir. A degron-İstikrarsızlaşmış GFP-(GFP-DGN) ve bir sabit GFP-dgnFS füzyon proteini oluşturulur ve bir lentiviral ifade vektörü kullanılarak hücre içine transdüksiyon ile uygulanır. Bu teknik, ubiquitin-proteasome etkinlik kolaylıkla Epifloresans ya da akış sitometrisi kullanılarak ölçülebilir hangi bir sabit GFP-dgn/GFP-dgnFS eksprese eden hücre soyu üretmek için olanak sağlar.

Yaşayan Hücreler ubikitin-proteozom Faaliyet Monitoring Degron (DGN)-destabilize Yeşil floresan proteini (GFP) tabanlı Muhabir Protein Kullanımı

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

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Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

Organik Solvent ve Asetik Asit olmadan Jeller Coomassie G-250 ile Proteinlerin Boyama

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

Sıvı Faz Kurtarma Moleküler Ağırlık Tabanlı Fraksiyonu GELFREE 8100 Fraksiyonu Sisteminin Kullanılması

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Helios Gen Tabancası kullanarak Soğan Hücrelerde Protein-Protein Etkileşimi Bimoleküler Floresan tamamlama (BiFC) Testi

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Embriyonik Kök Hücreler Yaşayan Kromatin Protein Dynamics Tedbir Tahliller (sıkı bağlamak ve FLIP) Photobleaching

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

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 ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Biyoparlaklık Rezonans Enerji Transferi kullanma Canlı Hücrelerde Protein-protein etkileşimleri araştıran

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Kültür Memeli Hücrelerde Biyolojik tanımında ubikitin ve Ubiquitin benzeri Sistemi Regülatörleri tanımak siRNA Eleme

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein Parçalanabilirliklerinin Sistematik Analizi muhabiri tabanlı Büyüme Deneyi

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Yaşayan Hücrelerde Protein-fragmanı tamamlama Assay (PCA) ile genom Protein-protein etkileşimi Eleme

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Membran proteinlerinin Yeşil floresan proteini temelli Ekspresyon elemesinde<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...