The binding reactions for the EMSA consisted of 1× EMSA buffer (0.01 mg/ml BSA, 0.1 mM DTT and 0.05 mM TCEP, 5% glycerol, 50 mM NaCl, 20 mM Tris pH 8) and unlabeled (50 nM–15 µM) protein (see figure legends for the exact protein and DNA concentration, and buffer conditions). Protein concentrations were prepared by 2-fold serial dilution. Samples were loaded onto either 10%, 12% or 4–15% pre-cast Mini-PROTEAN Tris-Glycine gel (TG; Bio-Rad) and electrophoresed for 25–45 min at 120 mV 4 °C in 1x TG buffer (Bio-Rad). EMSA experiments using unlabeled DNAs were stained with EtBr or SybrTM Green for 20 min prior to imaging. The gels were then imaged using ChemiDoc with the appropriate filters and analyzed through the Image Lab software (Bio-Rad). EMSAs were performed with 2–3 independent replicates.
Tg buffer
Manufactured by Bio-Rad
1x TG buffer is a commonly used buffer solution in molecular biology and biochemistry. It is a balanced salt solution that provides a suitable environment for various molecular biology techniques, such as gel electrophoresis and western blotting. The exact composition of the 1x TG buffer may vary depending on the specific application and manufacturer, but it typically contains a combination of tris and glycine as the primary buffering agents.
Automatically generated - may contain errors
2 protocols using tg buffer
1
EMSA Protocol for Protein-DNA Binding Analysis
2
Western Blot Protein Detection
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A total of 20 to 30 mg cell lysates were run on a precast gel (Bio-Rad, 12% SDS). Proteins were transferred to a PVDF membrane in transfer buffer [1x TG buffer (Bio-Rad), 20% methanol]. Membranes were blocked with 5% milk or BSA before incubation with primary antibody overnight at 4 C. Membranes were incubated with secondary antibodies (Dako) for 2 hours at room temperature, and bands were made visible by Pierce ECL Plus Substrate (Thermo).
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