The Large Volume His-Tag Labeling Kit RED-tris-NTA 2nd Generation provides convenient means for the site-specific, purification-free labeling of small amounts of His-tagged proteins with our fluorescent dye. This kit can be used for the labeling of any protein or peptide carrying a polyhistidine-tag (His-tag) and contains material sufficient for labeling for 3840 single-point TRIC/MST measurements. Labeling can be completed in 30 min, no removal of excess dye is required. The RED-tris-NTA 2nd Generation dye can bind efficiently to His-tags which contain at least six histidines with a Kd in the single digit nM range. By following this protocol, optimized for binding of RED-tris-NTA dye to His-tagged proteins eliminates an additional purification step and ensures the best results. RED-tris-NTA 2nd Generation dye shows fluorescence excitation and emission maxima at approximately 650 nm and 670 nm, respectively.