Unless stated otherwise, electrophoretic mobility shift assays (EMSA) contained 1 ng/ L NA templates (0.4 nM) in 20 L and were performed at 37 C in a binding buffer containing 20 mM Tris-HCl pH 7.4, 1 mM MgCl , 100 mM NaCl, and 1 mM DTT. NCps were diluted on ice in the reaction buffer. For reactions using NCp15, the buffers were supplemented with 0.1% (w/v) Tween 20. Reactions were initiated by addition of NC as appropriate and terminated at 30 min. (unless stated otherwise) by chilling the tubes on ice and addition of 10% (v/v) loading buffer (30% glycerol, 0.01% xylene cyanol, 10 mM Tris-HCl pH 7.4). Samples were then fractionated on 25 cm long, 1% (w/v) agarose gels (SeaKem LE Agarose, Lonza, Switzerland) in 0.5× TBE. Gels were run in a Sub-Cell GT cuvette (BioRad, USA) for 17–18 h at room temperature at 3 V/cm, stained with SybrGold (Invitrogen, USA), and scanned for fluorescence using a Typhoon 8600 (GE Healthcare, USA). All experiments were performed at least in triplicate.
Sub cell gt cuvette
Manufactured by Bio-Rad
Sourced in United States
The Sub-Cell GT cuvette is a laboratory equipment used for spectrophotometric analysis. It provides a contained environment for samples to be measured and analyzed using light absorption or emission techniques.
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2 protocols using sub cell gt cuvette
1
Electrophoretic Mobility Shift Assay for Nucleic Acid Binding
2
Quantifying DNA-Binding Affinity of Abf2p Mutants
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Two-fold serial dilutions of Abf2p and deletion mutants were performed on ice in 20 mM Tris–HCl pH 7.5 and 750 mM NaCl. Reactions were initiated by addition of proteins as appropriate and incubated for 30 min at 25°C. Double-stranded (ds) DNA binding experiments contained 2ng·μl−1 linearized M13mp18 and were performed in a binding buffer with a final composition of 20 mM Tris–HCl pH 7.5 and 100 mM NaCl (20μl final volume). Subsequently, samples were resolved on 25 cm long, 1.2% (w/v) agarose gels (SeaKem LE Agarose, Lonza) in 0.5× TBE (Sigma). Gels were run in a Sub-Cell GT cuvette (BioRad) for 17–18 h at room temperature at 3V/cm.The reactions with the four-way junctions contained 100 nM of the (25 bp × 4) DNA and the buffer was supplemented with 1mM MgCl2. After addition of a loading dye, the binding reactions were loaded on a 7% polyacrylamide gel (Sigma) in 0.5 × TBE. Gels were pre-run for 15 min and run at 11V/cm on a HoefferMiniVE cuvette placed on ice. Agarose and polyacrylamide gels were stained with SybrSafe (Molecular Probes) and scanned for fluorescence using a Typhoon 8600 scanner (GE Healthcare).
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