Name: rhoc gtpase activation assay
Uploaded: 2010-08-22T17:00:00
Description: Watch this Scientific Journal Video about RhoC GTPase Activation Assay at JoVE.com

Savunma Bölümü W81XWH-05-1-0005, W81XWH-06-1-0495, W81XWH-08-1-0029 ve W81XWH-08-1-0356

1. Hazırlayın ve GST-füzyon protein <ol><li> BamH1/EcoR1 pGEX3x vektör içine subcloned rhotekin Rho bağlayıcı etki alanı (RBD) ilk 90 N-terminal amino asitleri içeren JM109 yetkili bakterilerin Gliserol stokları yapılır. Yüksek verim sağlamak için aşağıdaki adımları, her bir deney için yapılmalıdır. Deneyimlerimize göre, taze GST-RBD sağlam ve doğru bir GTPaz aktivasyon tahlil için anahtar hazırlanmıştır. </li><li> Gliserol stokunun LB amp 50 ml (yaklaşık, Çözülme yok...

<ol>
	<li>van Golen, K. L., Wu, Z. F., Qiao, X. T., Bao, L. W., Merajver, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase,+a+novel+transforming+oncogene+for+human+mammary+epithelial+cells+that+partially+recapitulates+the+inflammatory+breast+cancer+phenotype.">RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype.</a> Cancer Res. 60 (20), 5832-5838 (2000).</li><li>van Golen, K. L., Wu, Z. F., Qiao, X. T., Bao, L., Merajver, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase+overexpression+modulates+induction+of+angiogenic+factors+in+breast+cells.">RhoC GTPase overexpression modulates induction of angiogenic factors in breast cells.</a> Neoplasia. 2 (5), 418-425 (2000).</li><li>Golen, K. L. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversion+of+RhoC+GTPase-induced+inflammatory+breast+cancer+phenotype+by+treatment+with+a+farnesyl+transferase+inhibitor.">Reversion of RhoC GTPase-induced inflammatory breast cancer phenotype by treatment with a farnesyl transferase inhibitor.</a> Mol Cancer Ther. 1 (8), 575-583 (2002).</li><li>Golen, K. L. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen+activated+protein+kinase+pathway+is+involved+in+RhoC+GTPase+induced+motility,+invasion+and+angiogenesis+in+inflammatory+breast+cancer.">Mitogen activated protein kinase pathway is involved in RhoC GTPase induced motility, invasion and angiogenesis in inflammatory breast cancer.</a> Clin Exp Metastasis. 19 (4), 301-311 (2002).</li><li>Yao, H., Dashner, E. J., van Golen, C. M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase+is+required+for+PC-3+prostate+cancer+cell+invasion+but+not+motility.">RhoC GTPase is required for PC-3 prostate cancer cell invasion but not motility.</a> Oncogene. 25 (16), 2285-2296 (2006).</li><li>Hall, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+Collagen+receptor+(alpha2beta1)+signaling+promotes+prostate+cancer+cell+invasion+through+RhoC+GTPase.">Type I Collagen receptor (alpha2beta1) signaling promotes prostate cancer cell invasion through RhoC GTPase.</a> Neoplasia. 10 (8), 797-803 (2008).</li><li>Sequeira, L., Dubyk, C. W., Riesenberger, T. A., Cooper, C. R., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+GTPases+in+PC-3+prostate+cancer+cell+morphology,+invasion+and+tumor+cell+diapadesis.">Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell diapadesis.</a> Clin Exp Metastasis. 25, 569-579 (2008).</li><li>van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL2+Induces+Prostate+Cancer+Transendothelial+Cell+Migration+via+Activation+of+the+Small+GTPase+Rac.">CCL2 Induces Prostate Cancer Transendothelial Cell Migration via Activation of the Small GTPase Rac.</a> J Cell Biochem. 104, 1587-1597 (2008).</li><li>Chatterjee, M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Int J Cancer. , (2010).</li><li>Sander, E. E., ten Klooster, J. P., van Delft, S., van der Kammen, R. A., Collard, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac+downregulates+Rho+activity:+reciprocal+balance+between+both+GTPases+determines+cellular+morphology+and+migratory+behavior.">Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior.</a> J Cell Biol. 147 (5), 1009-1022 (1999).</li></ol>

Prosedürün en kritik adım, taze GST-RBD nesil. Bu adımı yakın ilgi ödenmemesi başarısızlığın ana nedeni budur. GST-RBD (1.2) üretimi hücre lizatı (2.5) ile gecelik inkübasyon tüm adımları tek bir gün içinde yapılmalıdır. Bir diğer önemli adım, hücre lizatları üretmek için yeterli hücre vardır emin olun. RhoC GTPaz, hücre düşük seviyelerde bulunan olma eğilimindedir bakarken özel bir önem taşımaktadır.

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rhoc gtpase activation assay

RhoC GTPaz RhoA GTPaz% 91 homoloji vardır. Çünkü hücreleri yaygınlığı, RhoA GTPaz için birçok reaktifler ve teknikleri geliştirilmiştir. Ancak, RhoC GTPaz nispeten düşük seviyelerde metastatik kanser hücrelerinin ifade edilir. Bu nedenle, birkaç RhoC özel reaktifler geliştirilmiştir. Biz RhoC GTPaz tespit etmek için bir Rho aktivasyon tahlil adapte var. Bu teknik aktif RhoC GTPaz çekin bir GST-Rho bağlayıcı etki alanı füzyon proteini kullanır. Buna ek olarak, toplam düzeyleri (GTP ve GSYİH bağlı) RhoC GTPaz belirlemek için testin başında total protein hasat. Bu hücre aktif karşı toplam RhoC GTPaz belirlenmesi için sağlar. Ancak, bu prosedürün birçok ticari sürümleri geliştirilmiştir, ticari kitleri RhoA GTPaz için optimize edilmiştir ve genellikle RhoC GTPaz için iyi bir sonuç vermez. Testinin parçaları RhoC-spesifik antikor gelişimi yanı sıra modifiye edilmiştir.

Watch this Scientific Journal Video about RhoC GTPase Activation Assay at JoVE.com

RhoC GTPase Activation Assay

Bu protokol, hücre içinde aktif RhoC GTPaz düzeylerini belirlemek için tahlil aşağı çekme kullanır.

RhoC GTPaz Etkinleştirme Testi

RhoC GTPase has 91% homology to RhoA GTPase.  Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed.  ...

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