Name: intracellular refolding assay
Uploaded: 2012-01-24T13:00:00
Description: Watch this Scientific Journal Video about Intracellular Refolding Assay at JoVE.com

Danilo Maddalo er en modtager af et forsknings-fællesskab, som Young Investigator Group (YIG) fra Karlsruhe Institute of Technology.

1. Seeding cellerne <ol><li> Før du starter, varme op dyrkningsmediet, PBS 1X og trypsin i et vandbad ved 37 ˚ C </li><li> Tag de celler ud af kuvøsen og aspirér medium. </li><li> Forsigtigt på 5 mL PBS 1X på cellerne til at vaske dem. </li><li> Aspirer PBS 1X og anvende 1 mL trypsin indeholder 0,025% EDTA. </li><li> Drej forsigtigt pladen for at få en jævn fordeling af trypsin. </li><li> Placer celler i inkubator ved 37 ˚ C i 5-10 minutter (afhængig af celletype). Fra tid til anden kontrollere, om celler er...

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	<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>

I dette arbejde en protokol til måling af intracellulære hvorved man genfolder aktivitet molekylære anstandsdamer præsenteres. Hele analysen kan udføres i 3 til 4 dage som det fremgår af oversigten i Fig. 1. Robusthed og linearitet lyssignal produceret af ildflue og renilla luciferase repræsenterer en solid base for reproducerbarheden af ​​protokollen. Den kritiske trin i analysen er valget af en effektiv transfektion reagens for at si...

Til dette eksperiment en Luminometer form, Perkin Elmer &#39;1420 Luminescence Counter &#39;Vector Light&#39; blev brugt Programmer for Luminometer: Renilla: Pumpe 1, 100 μl injektion Reaction Buffer: Pump1, 70 μl injektion Luciferin: Pump2, 30μl indsprøjtning For de buffere forberede disse stamopløsninger med dobbeltdestilleret vand: <ul style="list-style:none"><li> - 1M MgSO 4: 246,48 g MgSO 4 * 7H 2 O i 1 liter </li><li> - 0,5 M EGTA: 190,18 g EGTA i 1 liter </li><li> - 1M KH 2 PO 4: 136,09 g KH 2 PO 4 i 1 liter </li><li> - 1M K2 HPO 4: 228,23 g K 2 HPO 4 * 3 H 2 O i 1 liter </li><li> - 5M NaCl: 292,2 g NaCl i 1 liter </li><li> - 0,5 M EDTA: 186,12 g EDTA * 2H 2 O i 1 liter &nbsp;</li></ul> Gly-Gly-buffer: <ul style="list-style:none"><li> - 25mm Glycylglycin </li><li> - 15mm MgSO 4 </li><li> - 4mm EGTA </li></ul> Til 1 liter brug 3,3 g af Glycylglycin, 15 ml af en 1M MgSO4 løsning og 8ml af en 0,5 M EGTA løsning. Løs i vand, pH indstilles til 7,8 og bringe til en samlet volumen på 1 liter. Reaktionsbuffer for Renilla / Coelenterazine buffer: <ul style="list-style:none"><li> - &amp; Nbsp; 13,4 mM KH 2 PO 4 </li><li> - 86,6 mM KH 2 PO 4 </li><li> - 0,5 M NaCl </li><li> - 1mm EDTA </li></ul> Til 1 liter brug 13,4 ml af en 1 M KH 2 PO 4-løsning,. 86,6 ml af en 1M K 2 HPO 4 løsning, 100ml af en 5M NaCl-opløsning og 2 ml af en 0,5 M EDTA-oploesning <table border="1"><tr><td></td><td> Firma </td><td> Katalog / Produkt nummer: </td></tr><tr><td> DMEM (Dulbecco er Modified Eagle Medium 1X) </td><td> GIBCO </td><td> 41966029 </td></tr><tr><td> Føtalt bovint serum Gold </td><td> PAA </td><td> A15-151 </td></tr><tr><td> X-treme Gene 9 DNA-transfektion Reagens </td><td> Roche </td><td&gt; 06365809001 </td></tr><tr><td> PBS Dulbecco er Fosfatbufferet Saline 1X </td><td> GIBCO </td><td> 14190-094 </td></tr><tr><td> MOPS 3 - (N-Morpholino) Propanesulfonic syre </td><td> SIGMA-ALDRICH </td><td> M1254 </td></tr><tr><td> Cycloheximid </td><td> SIGMA-ALDRICH </td><td> C7698 </td></tr><tr><td> Passiv lysisbuffer 5X </td><td> Promega </td><td> E194A </td></tr><tr><td> Glycylglycin EGTA MgSO 4 (MgSO 4 * 7H 2 O) </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 * 3 H 2 O) NaCl EDTA </td><td> Roth Roth Roth Roth </td><td> 3904,1 6878,2 3957,1 8043,1 </td></tr><tr><td> Luciferin 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 (adenosin trifosfat) </td><td> Roche </td><td> 10519987001 </td></tr></table>

intracellular refolding assay

Denne protokol beskriver en metode til at måle den enzymatiske aktivitet af molekylære anstandsdamer i en celle-baseret system og de mulige virkninger af stoffer med hæmmende / stimulerende aktivitet. Molekylær anstandsdamer er proteiner involveret i reguleringen af proteinfoldning 1 og spiller en afgørende rolle i at fremme cellernes overlevelse på stress fornærmelser som heat shock 2, næringsstof sult og eksponering for kemikalier / giftstoffer 3. Af denne grund anstandsdamer er fundet at være involveret i begivenheder som tumor udvikling, chemioresistance af kræftceller 4 samt neurodegeneration 5. Design af små molekyler i stand til at hæmme eller stimulere aktiviteten af disse enzymer er derfor en af de mest undersøgte strategier for kræftbehandling 7 og neurodegenerative sygdomme 9. Analysen her beskrevne giver mulighed for at måle hvorved man genfolder aktiviteten af ​​et bestemt molekylær anstandsdame og for at undersøge effekten af ​​compounds om sine aktiviteter. I denne metode gen af ​​den molekylære undersøgte anstandsdame er transficeret sammen med en vektor kodning for ildflue luciferase genet. Det er allerede blevet beskrevet, at denatureret ildflue luciferase kan refolded ved molekylær anstandsdamer 10,11. Som normalisering transfektion kontrol, er en vektor kodning for renilla luciferase genet transfekteret. Alle transfections beskrevet i denne protokol udføres med X-treme Gene 11 (Roche) i HEK-293 celler. I det første trin, er proteinsyntesen hæmmes ved at behandle cellerne med cycloheximid. Derefter protein udfoldning er foranlediget af heat shock ved 45 ° C i 30 minutter. Ved genopretning ved 37 ° C, er proteiner igen foldet ind i deres aktive kropsbygning og aktivitet ildflue luciferase bruges som read-out: jo mere lys vil blive produceret, jo mere protein vil have igen fået de oprindelige kropsbygning. Ikke-varme chokeret celler er sat som reference (100% af refoldedluciferase).

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

Intracellular Refolding Assay

I denne protokol en metode til at måle intracellulært protein, hvorved man genfolder efter heat shock er beskrevet. Denne metode kan bruges til at studere foldases som molekylær anstandsdamer og deres co-faktorer eller stoffer i stand til at påvirke deres aktivitet. Firefly luciferase aktiviteten bruges som reporter til at måle anstandsdame hvorved man genfolder aktivitet.

Intracellulære hvorved man genfolder Assay

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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