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Team:METU Turkey/Results Discussion/EMSA
From 2010.igem.org
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| - | <br>In order to determine protein-DNA interaction, we performed electrophoretic mobility shift assay or gel retardation assay. We used the proteins in two forms. The proteins used were reduced CooA and reduced -CO bounded CooA and also pCooF and pCooM were used as DNA components. Before starting experiment, we added DTT to inactive or oxidized CooA to reduce Ferric ion to ferrous ion in the CooA then we sparged carbonmonoxide (CO) to protein solution during 5 min. The DNA binding reaction was carried out by reaction buffer which did not contain reducing agents such as DTT and sodium dithionite. We had seven reaction for each EMSA experiment. These reactions are given below. | + | <br>In order to determine protein-DNA interaction, we performed electrophoretic mobility shift assay or gel retardation assay. We used the proteins in two forms. The proteins used were reduced CooA and reduced -CO bounded CooA and also pCooF and pCooM were used as DNA components. Before starting experiment, we added DTT to inactive or oxidized CooA to reduce Ferric ion to ferrous ion in the CooA then we sparged carbonmonoxide (CO) to protein solution during 5 min. The DNA binding reaction was carried out by reaction buffer which did not contain reducing agents such as DTT and sodium dithionite. We had seven reaction for each EMSA experiment. These reactions are given below.</br> |
| - | <br>Volume of each reaction was 40 ul. These samples included 20 ul 2X reaction buffer and 10 ul protein solutions and 10 ul DNA component solutions. After preparation the reaction mix, we incubated the samples at room temperature for DNA binding for 20 min. We run the samples at 100 V for 1.5 hours at 4C. Before running the 6% polyacrylamide gel added sodium dithionite that was sparged with Argon to provide anoxic condition, pre-run was done at 4C at 100 V for 45 min. After running for 1.5 hours, we stained the gel staining with Et-Br by shaking for 15 min to stain DNA. We analyzed the gel under UV and the result is obtained given below. | + | <br>Volume of each reaction was 40 ul. These samples included 20 ul 2X reaction buffer and 10 ul protein solutions and 10 ul DNA component solutions. After preparation the reaction mix, we incubated the samples at room temperature for DNA binding for 20 min. We run the samples at 100 V for 1.5 hours at 4C. Before running the 6% polyacrylamide gel added sodium dithionite that was sparged with Argon to provide anoxic condition, pre-run was done at 4C at 100 V for 45 min. After running for 1.5 hours, we stained the gel staining with Et-Br by shaking for 15 min to stain DNA. We analyzed the gel under UV and the result is obtained given below. </br> |
D9 | D9 | ||
| - | <br>There is a little retardation. When reduced -CO bounded CooA binds to pCooF , DNA retardation is expected, however gel image said that the retardation happened when compared to other pCooF /pCooM - CooA binding. | + | <br>There is a little retardation. When reduced -CO bounded CooA binds to pCooF , DNA retardation is expected, however gel image said that the retardation happened when compared to other pCooF /pCooM - CooA binding.</br> |
Revision as of 00:09, 28 October 2010
In order to determine protein-DNA interaction, we performed electrophoretic mobility shift assay or gel retardation assay. We used the proteins in two forms. The proteins used were reduced CooA and reduced -CO bounded CooA and also pCooF and pCooM were used as DNA components. Before starting experiment, we added DTT to inactive or oxidized CooA to reduce Ferric ion to ferrous ion in the CooA then we sparged carbonmonoxide (CO) to protein solution during 5 min. The DNA binding reaction was carried out by reaction buffer which did not contain reducing agents such as DTT and sodium dithionite. We had seven reaction for each EMSA experiment. These reactions are given below.
Volume of each reaction was 40 ul. These samples included 20 ul 2X reaction buffer and 10 ul protein solutions and 10 ul DNA component solutions. After preparation the reaction mix, we incubated the samples at room temperature for DNA binding for 20 min. We run the samples at 100 V for 1.5 hours at 4C. Before running the 6% polyacrylamide gel added sodium dithionite that was sparged with Argon to provide anoxic condition, pre-run was done at 4C at 100 V for 45 min. After running for 1.5 hours, we stained the gel staining with Et-Br by shaking for 15 min to stain DNA. We analyzed the gel under UV and the result is obtained given below. D9
There is a little retardation. When reduced -CO bounded CooA binds to pCooF , DNA retardation is expected, however gel image said that the retardation happened when compared to other pCooF /pCooM - CooA binding.
