Figure 3.

The GAP-tag can be used to introduce site-specific modifications with NAD⁺ analogs

(A) Site-specific biotinylation of the GAP-tag. GAP-tagged YFP was mixed with NAD⁺ or 6-biotin-17-NAD⁺ in absence or presence of PtxS1. The reactions were blotted on a nitrocellulose membrane, and detection of biotin was done with streptavidin-HRP.

(B) YFP-GAP was MARylated with PtxS1 using NAD⁺ or 6-propargyladenine-NAD⁺ containing an alkyne group. The resulting proteins YFP-GAP(MAR) or YFP-GAP(MAR-alkyne) or buffer were mixed with Cy3-azide or Cy5-azide, and the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction was performed by addition of 5 mM sodium ascorbate, 300 μM CuSO₄, and 600 μM L-histidine. The samples were incubated for 3 h at room temperature and blotted on nitrocellulose membranes, and visible-light images were taken. Unreacted Cy3-azide or Cy5-azide was removed by washing of the membranes in Tris-buffered saline/Tween-20. Visible-light and fluorescent images were taken.