Nt 647 nhs dye

20 μM protein was labeled with a two-fold excess of NT-647-NHS dye (NanoTemper Technologies, Munich Germany), following the manufacturer’s instructions. Protein had a degree of labeling of 1 dye molecule per protein molecule. Binding affinity of proteins for ligands was determined using Microscale Thermophoresis (MST) with a Monolith NT.115 instrument (NanoTemper Technologies, Germany). 10 μL of 20 nM labelled protein was mixed with 10 μL of ligand in 40 mM citrate-phosphate, 250 mM NaCl, 0.05% Tween-20 pH 7.4 or 5.4 μL of protein-ligand mixture was loaded into “Premium Grade Capillaries” (NanoTemper Technologies) and thermophoresis was measured at 22 °C for 22 s with 40% LED power and medium thermophoresis power. Data from three independent measurements were combined and analysed using the MO.Affinity Analysis software version 2.1 (NanoTemper Technologies), fitted to a single binding site model (Eq. 1) where U is the unbound normalised fluorescence, B is the fully bound fluorescence, [l] is concentration of ligand. Data were plotted using Igor Pro version 7.05 (Wavemetrics Inc., USA). $$f(l)=\mathrm{U}+\frac{(\mathrm{B}-\mathrm{U})\times [l]+[\mathrm{HtxB}]+K_{\mathrm{d}}-\sqrt{{([l]+[\mathrm{HtxB}]+K_{\mathrm{d}})}^2-4\times [l]\times [\mathrm{HtxB}]}}{2\times [\mathrm{HtxB}]}$$

MST was performed on a Monilith NT.115 system (Nano Temper Technologies, DE). 6× His IDOL was labelled with NT-647-NHS dye (Nano Temper Technologies, DE) as per manufacturer's instruction and used at a final concentration of 50 nM throughout the assays. Peptides were subject to dilution into assay buffer (10 mM HEPES, 300 mM NaCl, 1 mM TCEP, 0.05% TWEEN-20, pH 8.0) with a final assay concentration of 5% DMSO. Solutions containing peptide and labelled IDOL were loaded in to premium capillaries (Nano Temper Technologies, DE) for measurement.

For the purpose of MST measurements, DSS1 was labelled by NT-647-NHS dye according to the manufacturer protocol (NanoTemper Technologies). Constant concentration of labelled DSS1 protein (50 nM) was mixed with increasing concentrations of RAD52 (1 nM to 36 μM) in Tris-Acetate buffer (30 mM Tris-acetate pH 7.5, 1 mM DTT) in a series of 16 independent capillaries. Mixed proteins were incubated at 25°C for 10 min and measured using low MST power and 20% of Excitation power. Changes in fluorescence were plotted against RAD52 concentration and K_d value was calculated using quadratic binding equation for six independent measurements at two different MST power.