Seakem gtg agarose solution

An EMSA was performed by incubating proteins with Cy5 or fluorescein-labeled molecules [final concentrations of 100 nM] in anisotropy buffer to a final volume of 11 μL at room temperature for 1 h. After incubation, 1.3 μL of ice-cold 50% glycerol was added to the mix. The complexes were separated from unbound species by electrophoresis on a 0.7% nondenaturing gel using a SEAKEM GTG agarose solution (Lonza) made with 1× TBE buffer. Samples were separated at 4 °C in 1× TBE buffer for 1.5 h at a 66 V constant voltage. The gels were visualized by scanning with a FLA9500 Typhoon instrument using the Cy2 or Cy5 excitation laser at a 600 PMT voltage and 50 μm resolution. In cases where concentrations are not explicit, 500 nM PABP to 100 nM poly(A) RNA ratio was used with 5 μM NS1 when excess NS1 was used. Gels were analyzed and quantified with ImageJ software.^{20 (link)}

An EMSA was performed by incubating fluorescein-labeled RNA [final concentrations of 100 nM] with protein in anisotropy buffer to a final volume of 11 μL at room temperature for 1 h. After incubation, 1.3 μL of ice-cold 50% (v/v) glycerol and xylene cyanol were added to the mix. The complexes were separated from unbound species by electrophoresis on a 0.7% nondenaturing gel using a SEAKEM GTG agarose solution (Lonza) made with 1× TBE buffer. Samples were separated at 4 °C in 1× TBE buffer for 1.5 h at a 66 V constant voltage. The gels were visualized by scanning with a FLA9500 Typhoon instrument using the Cy2 excitation laser at a 600 PMT voltage and 50 μm resolution. In cases where concentrations are not explicit, 500 nM PABP to 100 nM poly(A) RNA ratio was used. Gels were analyzed with ImageJ software [74 (link)].