Biomod/2011/Slovenia/BioNanoWizards/methbret

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BRET assay


BRET DNA target containing 2C7 and AZPA4 zinc finger binding sites separated by 2 bp spacer was annealed by first heating the sample containing oligonucleotides at 100 μM concentration in MQ and gradually cooled down in 1 °C / min steps. The 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). Protein samples were prepared as follows: isolated proteins at concentrations of ~5 μM in case of MBP-YFP-AZPA4 and ~0.5 μM in case of MBP-RLuc-2C7 were diluted to final concentration of 100 nM. DNA target was added to a final concentration of 50 nM. 2-fold excess of the purified BRET partner fusions was used to prevent the situation where DNA target would be occupied by only one of the protein partners. Samples were pipetted into a Perkin Elmer OptiPlate 96-well white plate and incubated for 3 h at 4 °C. BRET measurements were performed on 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 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 directly compared. The protocol for BRET measurement consisted of the steps listed:

  1. Dispense step during which 100 μL of the coelenterazine substrate was injected into the well
  2. Shake step for 5 seconds
  3. Luminescence measurement with 460 DF50 emission filter with 1 sec counting time
  4. Luminescence measurement with F535 (FITC Fluorescein) with 1 sec counting time
 

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