Additional file 8: Figure S5. of Sequence-based prediction of permissive stretches for internal protein tagging and knockdown

Systematic identification of ATP sinks in CFX. CFX was fractionated by step-wise (increments of 10% per step) ammonium sulphate precipitation. Fractions were separated by native PAGE. The gel was subsequently incubated in ATP and stained to detect liberated inorganic phosphate (Pi) by malachite green as described [67] (lower panel). In brief: The gel was dipped for 30 min in redox buffer (30 mM Tris/HCl, 80 mM KCl, 5 mM MgCl2 10 mM DTT), followed by 1 h incubation in 10 mL substrate buffer (30 mM Tris, 80 mM KCl, 5 mM MgCl2, 10 mM ATP) at 37 °C. We added 2 mL of a malachite green solution (1.2 mL 0.44 mg malachite green in 100 mL H2O/H2SO4 and 0.8 mL 7.5% ammonium molybdate) directly to the substrate buffer. Colour development was allowed to proceed for 1 h. Samples were split after fractionation and additionally with Coomassie Blue to visualise all present protein bands (upper panel). Dark green spots indicate the presence of ATPase activity. Spots were cut from the gel and proteins were identified by mass spectrometry. White arrows spots corresponding to GroEL, red arrows spots corresponding to the F1 part of ATP synthase grey arrow spot corresponding to DnaK; purified GroEL was used as a positive control (C). (PNG 344 kb)