Polyethylene imine TLC resolves [32P] products. The structure of AppA is shown to the right of its label. The lane marked “0” is an unincubated complete reaction, “inc” is the untemplated reaction after 19 h at 12°C, and “inc + GppG” implies the same incubation but with 10 mM GppG added. This is the chromatographic system previously used for detection of AppA (Puthenvedu et al. 2015), and the identity of the product has been confirmed by HPLC (D Puthenvedu and M Yarus, in prep.). By determining the fraction of total radioactivity in the product spot, product concentration can be calculated from known total [pA]. The bar marked pApA at the lower right of the chromatogram shows the position of 2′–5′ pApA (top of the bar) and 3′–5′ pApA (bottom), if present.
