Neslab rte7 water bath

The equilibrium binding of RBM7 RRM to RNA was characterized by fluorescence anisotropy. The RNA was labeled at 5′ end with fluorescein fluorophore. The fluorescein was excited at 488 nm and its emission was collected at 520 nm. The width of both excitation and emission monochromatic slits was varying from 9 to 14 nm depending on measured RNA sequence. Integration time was set to 3 s. All measurements were conducted on a FluoroMax-4 spectrofluorometer (Horiba Jobin-Yvon). The instrument was equipped with a heated cell holder with a Neslab RTE7 water bath (Thermo Scientific). The system was operated by FluorEssence software (version 2.5.3.0 and V3.5, Horiba Jobin-Yvon). All measurements were performed at 20°C in 50 mM Tris 7.0 buffer supplemented with 200 mM sodium chloride and 10 mM 2-mercaptoethanol (pH 8). Ten nanomoles of RNA (in volume of 1.4 ml) was titrated with increasing amounts of RBM7 RRM protein sample (in the same buffer). Each data point in plot is an average of three measurements. The data were analyzed using Gnuplot (version 4.4.3) and Xmgrace (version 5.1.16). The data were normalized for visualization purposes and the experimental isotherms were fit to a single-site binding model according to Heyduk and Lee using non-linear least squares regression.

The equilibrium binding of Rtt103p CID constructs to differently phosphorylated CTD was analysed by fluorescence anisotropy. The CTD peptides were N‐terminally labelled with the 5,6‐carboxyfluorescein (FAM). The measurements were conducted on a FluoroLog‐3 spectrofluorometer (Horiba Jobin‐Yvon Edison, NJ). The instrument was equipped with a thermostatted cell holder with a Neslab RTE7 water bath (Thermo Scientific). Samples were excited with vertically polarized light at 467 nm, and both vertical and horizontal emissions were recorded at 516 nm. All measurements were conducted at 10°C in 35 mM KH₂PO₄, 100 mM KCl (pH 6.8). Each data point is an average of three measurements. The experimental binding isotherms were analysed by DynaFit using 1:1 model with non‐specific binding 39.

Nrd1 CID and its mutants were produced and purified as described previously (Kubicek et al, 2012). The equilibrium binding of the different versions of Nrd1 CID to Trf4 NIM and Sen1 NIM was analysed by FA. The NIM peptides were N‐terminally labelled with the 5,6‐carboxyfluorescein (FAM). The measurements were conducted on a FluoroLog‐3 spectrofluorometer (Horiba Jobin‐Yvon Edison, NJ). The instrument was equipped with a thermostated cell holder with a Neslab RTE7 water bath (Thermo Scientific). Samples were excited with vertically polarized light at 467 nm, and both vertical and horizontal emissions were recorded at 516 nm. All measurements were conducted at 10°C in 50 mM Na₂HPO₄ 100 mM NaCl pH = 8. Each data point is an average of three measurements. The experimental binding isotherms were analysed by DynaFit using 1:1 model with non‐specific binding (Kuzmic, 2009).