Fig. 5

One-step affinity purifications using ALFA Selector resins. a, b E. coli (a) or HeLa (b) lysates blended with purified ALFA-tagged shGFP2 (a, left lane) were incubated with ALFA Selector^ST, ALFA Selector^PE or an analogous resin without immobilized sdAb (Selector Control). After washing with PBS, the resins were incubated with 200 µM ALFA peptide for 20 min (E1–peptide) before eluting remaining proteins with SDS sample buffer (E2–SDS). Indicated fractions were analyzed by SDS–PAGE and Coomassie staining. Eluate fractions correspond to the material eluted from 1 µL of resin. c Native pull-down of an E. coli inner membrane protein complex. Left: Sketch of the target protein complex. Right: Detergent-treated lysates from a ΔyfgM strain complemented with either C-terminally ALFA-tagged (left panel) or untagged YfgM (right) were incubated with ALFA Selector^PE. After washing with PBS, bound proteins were eluted using 200 µM ALFA peptide. Samples corresponding to 1/800 of the input and non-bound material or 1/80 of eluate fractions were resolved by SDS page and analyzed by Western blot. ALFA Selector^PE specifically immunoprecipitated the native protein complex comprising ALFA-tagged YfgM and its interaction partner PpiD. In the control reaction (no ALFA-tag on YfgM), both proteins were absent in the eluate. Complete blots in Supplementary Fig. 8. Color code used in sketch: PpiD (purple), YfgM (pink)