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| <div class="entry"><big><big><big><big><span | | <div class="entry"><big><big><big><big><span |
| style="color: black; font-weight: bold;">BRET assay</span></big></big></big></big><br> | | style="color: black; font-weight: bold;">BRET assay</span></big></big></big></big><br> |
| <br> | | <br><br> |
| <span style="font-family: Arial;"> | | <span style="font-family: Arial;"> |
| BRET DNA target bearing 2C7 and AZPA4 zinc finger binding sites | | BRET DNA target containing 2C7 and AZPA4 zinc finger binding sites |
| separated by 2 bp was annealed by first heating the sample containing | | separated by 2 bp spacer was annealed by first heating the sample containing |
| oligonucleotides in MQ at 100 μM concentration and then gradually | | oligonucleotides at 100 μM concentration in MQ and gradually |
| cooled down in 1 °C / min steps. Then annealed target was diluted to 50 | | 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, | | μM with 2x buffer (20 mM Tris-HCl, 300 mM NaCl, 2 mM DTT, 20 mM MgCl<sub>2</sub>, |
| pH 8,0). The samples were prepared as follows: isolated proteins with | | 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 | | 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 | | 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 |
| 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 | | 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 | | of the protein partners. Samples were pipetted into a Perkin Elmer OptiPlate |
| 96-well white plate and incubated for 3 hours at 4 °C. BRET | | 96-well white plate and incubated for 3 h at 4 °C. BRET |
| measurements were taken using Berthold's Mithras LB940 Multimode | | measurements were performed on Berthold's Mithras LB940 Multimode |
| Microplate Reader. The instrument's injector was washed with MQ prior | | Microplate Reader. The instrument's injector was washed with MQ prior |
| the use and primed with buffer containing Renilla luciferase's | | the use and primed with buffer containing Renilla luciferase |
| substrate coelenterazine h (Synchem) diluted to a final concentration | | substrate coelenterazine h (Synchem) diluted to a final concentration |
| of 8 μM. Coelenterazine h was injected to each well separately so that | | of 8 μM. Coelenterazine h was injected to each well separately so that |
| the photon count between samples could be compared. The protocol for | | the photon count between samples could be directly compared. The protocol for |
| BRET measurement consisted of the steps listed: | | BRET measurement consisted of the steps listed: |
| </span><br style="font-family: Arial;"> | | </span><br style="font-family: Arial;"> |
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<body>
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:
- Dispense step during which 100 μL of the coelenterazine
substrate was injected into the well
- Shake step for 5 seconds
- Luminescence measurement with 460 DF50 emission filter with
1 sec counting time
- Luminescence measurement with F535 (FITC Fluorescein) with
1 sec counting time
</body>
</html>
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