Regeneration of Spy&Go resin

Lysate from bacterial expression of SpyTag-MBP was purified using 0.5 mL Spy&Go resin as above. The resin was equilibrated with 2 × 10 packed resin volumes of TP buffer before regeneration. Spy&Go resin was then regenerated at 25 °C by incubating with 3 × 10 packed resin volumes of 4 M imidazole in TP buffer pH 7.0 for 5 min each time, washing with 1 × TP buffer, incubating with 3 × 10 packed resin volumes of 6 M guanidine hydrochloride pH 2.0 for 5 min each time, washing with 1 × TP buffer, incubating with 3 × 10 packed resin volumes of 0.1 M NaOH for 1 min each time, and washing with 2 × TP buffer before storage in 20% (v/v) ethanol in MilliQ water at 4 °C. Supernatant from each stage was analyzed by SDS-PAGE with Coomassie staining. New resin, resin pre-regeneration and regenerated resin were also analyzed by boiling in SDS-PAGE loading buffer, followed by SDS-PAGE with Coomassie staining. Lysate from bacterial expression of αDR5-SpyTag was added to new resin or regenerated resin and purified using the standard Spy&Go protocol, before analysis by SDS-PAGE with Coomassie staining. To test capture after storage, Spy&Go resin was stored in 20% (v/v) ethanol for 12 weeks at at 4 °C, before purification of SpyTag-MBP from bacterial lysate following the standard protocol.