Electrophoresis mobility shift assay Kit (Thermo Fisher Scientific) was used to detect binding of NbERF-IX-33a to the NbEAS4 promoter. The DNA fragment of NbEAS4 promoter (30 ng) and MBP or MBP- NbERF-IX-33a proteins (1 μg) were mixed and incubated in 10 μl binding buffer [125 mM HEPES-KOH, 20 mM KCl, 0.5 mM EDTA, 50% (v/v) glycerol, 5 mM DTT, pH7.5] for 20 min at 4°C. 2 μl of 6× loading buffer was added to stop the reaction. The complexes were resolved on 6% non-denaturing polyacrylamide gels (Thermo Fisher Scientific) and stained with SYBR green dye.
Non denaturing polyacrylamide gels
Manufactured by Thermo Fisher Scientific
6% non-denaturing polyacrylamide gels are laboratory equipment used for electrophoresis, a technique that separates biomolecules based on their size and charge. These gels are composed of 6% polyacrylamide and are designed to maintain the native structure of the biomolecules during the separation process without denaturing them.
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Lab products found in correlation
2 protocols using non denaturing polyacrylamide gels
1
Investigating Transcription Factor Binding
2
Binding Assay of NbERF-IX-33a to NbEAS4 Promoter
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Electrophoresis mobility shift assay Kit (Thermo Fisher Scientific) was used to detect binding of NbERF-IX-33a to the NbEAS4 promoter. The DNA fragment of NbEAS4 promoter (30 ng) and MBP or MBP-NbERF-IX-33a proteins (1 µg) were mixed and incubated in 10 µl binding buffer [125 mM HEPES-KOH, 20 mM KCl, 0.5 mM EDTA, 50％ (v/v) glycerol, 5 mM DTT, pH7.5] for 20 min at 4˚C. 2 µl of 6 x loading buffer was added to stop the reaction. The complexes were resolved on 6% non-denaturing polyacrylamide gels (Thermo Fisher Scientific) and stained with SYBR green dye.
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