His tag labeling kit red tris nta

Microscale thermophoresis (MST) experiments were performed using a Monolith NT.115 instrument (NanoTemper). All experiments were performed using SEC purified Taf14 proteins (His-Taf14_FL, His-Taf14_ET, His-Taf14_YEATS, or Taf14_ET) in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0–0.2 mM TCEP. For the Taf14_ET:Taf2_CT interaction, the concentration of fluorophore, FAM-labeled Taf2_CT peptide (Synpeptide, 1392–1703 aa), was 20 nM. To monitor binding between Taf2_CT and Taf14_FL or Taf14_YEATS, and Taf14_Linker and Taf14_ET, His-tagged proteins were labeled using a His-Tag Labeling Kit RED-tris-NTA (2^nd Generation, Nanotemper), and the concentration of each labeled protein was kept at 20 nM. Dissociation constants were determined using a direct binding assay in which increasing amounts of unlabeled binding partner (Taf2_CT peptide or Taf14_Linker peptide) were added stepwise. The measurements were performed at 80% LED and medium MST power with 3 s steady state, up to 20 s laser on time and 1 s off time. The K_d values were calculated using MO Affinity Analysis software (NanoTemper) (n = 2 or 3). Figures were generated in GraphPad PRISM.

The antigen labeling was carried out according to the instructions in the His-Tag labeling kit Red-tris-NTA (Nanotemper, München, Germany).
Her2-His (Her2/ERBB2 Protein, Human, Recombinant (ECD, His Tag), sinoBiological) was diluted to 200 nM in PBST buffer (137 mM NaCl, 2.5 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4, 0.05% Tween-20). Tris-NTA dyes were diluted in PBST buffer to a final concentration of 100 nM. A 100 µL volume of protein was then mixed with 100 µL of dye, and the reaction mixtures were incubated for 30 min at room temperature in the dark and then centrifuged for 10 min at 10,000× g. The labeling was verified by following the instructions of the pretest of the MO.Control software (Nanotemper, München, Germany).
For the MST binding experiment, the concentration of fragments was diluted to 2 µM in PBS. This solution was used for a 1:1 serial dilution using 16 dilution steps, with a final volume of 6 µL for each point of the dilution series. Afterwards, 6 µL of HER2-Dye was added to all steps of the dilution series, giving a final ligand concentration of 5 nM. The reaction was incubated for 30 min at room temperature and loaded into Monolith NT.115 MST Premium Capillaries. The MST experiment was carried out using 100% LED power and medium MST power for the NT.115 RED instrument (Nanotemper, München, Germany).

Oligonucleotides (ODNs) were obtained from Litekh, Moscow, Russia (purity ≥ 95%, HPLC). Recombinant mouse CTCF with polyhistidine (His) tag was purchased from MyBioSource, San Diego, CA, USA. Recombinant human HMGB2 with His tag was purchased from Abcam, Cambridge, UK. Recombinant human HMGN1 and HMGN3 with His tags were purchased from LifeSpan BioSciences, Seattle, WA, USA. For microscale therophoresis (MST) assays, the proteins were labeled with the His-Tag Labeling Kit RED-tris-NTA (NanoTemper Technologies, Munich, Germany) according to the manufacturer’s protocol. G4 ligands pyridostatin (PDS) and bisquinolinium-derivatized phenanthroline-dicarboxamide (Phen DC3) were obtained from Sigma-Aldrich (St. Louis, MO, USA).