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 is a recipient of a research fellowship as Young Investigator Group (YIG) from the Karlsruhe Institute of Technology.

1. Seeding the cells

<ol> 
 <li>Before starting, warm up the culture medium, the PBS 1X and the trypsin in a water bath at 37&#730;C</li>
 <li>Take the cells out of the incubator and aspirate the medium.</li>
 <li>Gently apply 5 mL of PBS 1X on the cells to wash them.</li>
 <li>Aspirate the PBS 1X and apply 1 mL of trypsin containing 0,025% EDTA.</li>
 <li>Gently rotate the plate to have an even distribution of the trypsin.</li>
 <li>Place the cells in the incubator at 37&#730;C for 5-10 minutes (...

<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>

In this work a protocol to measure intracellular refolding activity of molecular chaperones is presented. The whole assay can be performed in 3 to 4 days as shown by the overview in Fig. 1.

The robustness and linearity of the light signal produced by the firefly and the renilla luciferase represent a solid base for the reproducibility of the protocol. 

The critical step of the assay is the choice of an efficient transfection reagent to ensure the ...

For this experiment a Luminometer form Perkin Elmer '1420 Luminescence Counter' Vector Light&prime; was used

Programs for the Luminometer:

Renilla: Pump 1, 100 &mu;l injection

Reaction Buffer: Pump1, 70 &mu;l injection

Luciferin: Pump2, 30&mu;l injection


For the buffers prepare these stock solutions in double distilled water:

<ul style="list-style:none">
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1M MgSO4 : 246,48g MgSO4*7H2O in 1 liter</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0,5M EGTA: 190,18g EGTA in 1 liter</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1M KH2PO4: 136,09g KH2PO4 in 1 liter</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1M K2HPO4: 228,23g K2HPO4*3H2O in 1 liter</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5M NaCl: 292,2g NaCl in 1 liter</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0,5M EDTA: 186,12g EDTA*2H2O in 1 liter&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;</li>
</ul>

GLY-GLY-buffer: 

<ul style="list-style:none">
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;25mM Glycylglycin </li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;15mM MgSO4</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4mM EGTA</li>
</ul>

For 1 liter use 3,3g of Glycylglycin, 15ml of a 1M MgSO4 solution and 8ml of a 0,5M EGTA solution. Solve in water, adjust the pH to 7,8 and bring to a total volume of 1 liter.


Reaction buffer for Renilla/Coelenterazine buffer: 

<ul style="list-style:none">
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;13,4mM KH2PO4</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;86,6mM KH2PO4</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0,5M NaCl</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1mM EDTA</li>
</ul>

For 1 liter use 13,4ml of a 1M KH2PO4 solution, 86,6ml of a 1M K2HPO4 solution, 100ml of a 5M NaCl solution and 2ml of a 0,5M EDTA solution. 
<table border="1">
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalog/Product Number:</td>
 </tr>
 <tr>
 <td>DMEM (Dulbecco&rsquo;s Modified Eagle Medium 1X)</td>
 <td>GIBCO</td>
 <td>41966029</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum Gold</td>
 <td>PAA</td>
 <td>A15-151</td>
 </tr>
 <tr>
 <td>X-treme Gene 9 
 DNA Transfection Reagent</td>
 <td>Roche</td>
 <td>06365809001</td>
 </tr>
 <tr>
 <td>PBS 
 Dulbecco&rsquo;s Phosphate Buffered Saline 1X</td>
 <td>GIBCO</td>
 <td>14190-094</td>
 </tr>
 <tr>
 <td>MOPS 
 3-(N-Morpholino)Propanesulfonic acid</td>
 <td>SIGMA-ALDRICH</td>
 <td>M1254</td>
 </tr>
 <tr>
 <td>Cycloheximide</td>
 <td>SIGMA-ALDRICH</td>
 <td>C7698</td>
 </tr>
 <tr>
 <td>Passive Lysis Buffer 5X</td>
 <td>Promega</td>
 <td>E194A</td>
 </tr>
 <tr>
 <td>
 Glycylglycin 
 EGTA 
 MgSO4 (MgSO4*7H2O)</td>
 <td>SIGMA-ALDRICH 
 Roth 
 Roth</td>
 <td>G7278 
 3054.2 
 P027.1 </td>
 </tr>
 <tr>
 <td>KH2PO4 
 K2HPO4 (K2HPO4*3H2O) 
 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 (Adenosine Triphosphate)</td>
 <td>Roche</td>
 <td>10519987001</td>
 </tr>
</table>

intracellular refolding 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 with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding1 and have a crucial role in promoting cell survival upon stress insults like heat shock2, nutrient starvation and exposure to chemicals/poisons3. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells4 as well as neurodegeneration5. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy7 and neurodegenerative disorders9. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene 11 (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45&deg;C for 30 minutes. Upon recovery at 37&deg;C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).

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

Intracellular Refolding Assay

In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.

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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