Monolith his tag labeling kit red tris nta 2nd generation kit

Recombinant Human TLR4 (R&D Systems) was re-suspended in a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, and 1 mM BME. TLR4 was fluorescently labeled using the NanoTemper Monolith His-Tag Labeling Kit RED-tris-NTA 2nd Generation kit (MO-L018) per the manufacturer's protocol. TLR4 was titrated with recombinant MBL (clinical grade) with concentrations varying from 20.8 μM to 635 pM, while keeping the labeled TLR4 at 5 nM. Samples were loaded into standard capillaries, and Microscale thermophoresis (MST) experiments were carried out using a Monolith NT.115 pico (NanoTemper Technologies GmbH, Munich, Germany). The thermophoresis was monitored at 20% LED power and high MST power. Data were analyzed and fit to the Kd model using MO. Affinity software (version 2.3) (NanoTempet Technologies, CA) in duplicate.

The recombinant protein hRAD51
was labeled with the Monolith His-Tag labeling kit RED-tris-NTA 2nd
Generation kit (NanoTemper Technologies). MST measurements were simultaneously
performed on 16 capillaries containing a constant concentration (25
nM) of labeled RED-tris-NTA 2nd Generation His-hRAD51 protein and
16 different concentrations of 35d in order to determine
a concentration-dependent MST binding curve. The highest 35d concentration tested was 40 μM. Measurements were carried
out in MST buffer (20 mM Hepes (pH 8.00), 250 mM KCl, 0.1 mM EDTA,
5% (v/v) glycerol, 5% DMSO).