Biomod/2011/Slovenia/BioNanoWizards/methbret

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</ul> <!-- End PureCSSMenu.com MENU --> </div> </div> </div> <!-- end #header --> <div id="page" class="container"> <div id="content"> <div class="post"> <div class="entry"><big><big><big><big><span 

style="color: black; font-weight: bold;">BRET assay</span></big></big></big></big><br>

<br> <span style="font-family: Arial;"> BRET DNA target bearing 2C7 and AZPA4 zinc finger binding sites separated by 2 bp was annealed by first heating the sample containing oligonucleotides in MQ at 100 μM concentration and then gradually cooled down in 1 °C / min steps. Then annealed target was diluted to 50 μM with 2x buffer (20 mM Tris-HCl, 300 mM NaCl, 2 mM DTT, 20 mM MgCl2, pH 8,0). The samples were prepared as follows: isolated proteins with concentrations of ~5 μM in case of MBP-YFP-AZPA4 and ~0.5 μM in case of MBP-RLuc-2C7 were diluted to a final concentration of 100 nM. In case of a BRET sample DNA target was added to a final concentration of 50 nM. 2-fold excess of the purified BRET partner fusions was used so as to prevent the situation where DNA target would be occupied by only one of the proteins. The samples were pipetted in a Perkin Elmer OptiPlate 96-well white plate and incubated for 3 hours at 4 °C. BRET measurements were taken using Berthold's Mithras LB940 Multimode Microplate Reader. The instrument's injector was washed with MQ prior the use and primed with buffer containing Renilla luciferase's substrate coelenterazine h (Synchem) diluted to a final concentration of 8 μM. Coelenterazine h was injected to each well separately so that the photon count between samples could be compared. The protocol for BRET measurement consisted of the steps listed: </span><br style="font-family: Arial;"> <br> <ol> 

 <li>Dispense step during which 100 μL of the coelenterazine

substrate was injected into the well</li> 

 <li>Shake step for 5 seconds</li>
 <li>Luminescence measurement with 460 DF50 emission filter with

1 sec counting time</li> 

 <li>Luminescence measurement with F535 (FITC Fluorescein) with

1 sec counting time</li> </ol> <!-- TU SE KONEA GLAVNO BESEDILO NA STRANI --> </div> </div> </div> <!-- end #content --> <div style="clear: both;">&nbsp;</div> </div> <!-- end #page --></div> <div id="footer-content" class="container"> <div id="footer-bg"> <table 

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