Bacterial strains and plasmids
Media and antibiotics
SILAC labeling
Cell lysis and protein extraction
Protein digestion in solution
Phosphopeptide enrichment
High-pH reversed-phase peptide fractionation on commercial spin columns
Offline high-pH reversed-phase peptide fractionation
SDS–polyacrylamide gel electrophoresis and in-gel digestion
Incorporation and mixing check
Protein purification by StageTips
LC-MS/MS measurement
MS data processing and analysis
His-tag affinity purification
In vitro kinase assay measured by MS
Dynamics of HipA and HipA7 in vitro (auto)phosphorylation measured by autoradiography
Determination of cell viability on SMG plates
Measurement of persistence
His6-HipA and His6-HipA7. Plasmid pBAD33::6his hipA was transformed into MG1655 strain. An overnight culture was grown in LB medium containing chloramphenicol (25 μg/ml) and 0.4% glucose, washed, diluted 1000× into 2 liters of LB medium containing chloramphenicol (25 μg/ml), and grown at 37°C. The expression of hipA and hipA7 was induced at OD600nm of 0.4 with 0.2% arabinose for 2 hours. His6-HipA7 was produced from pBAD33::6his hipA7 in 1 liter of LB medium in the same way as His6-HipA.
His6-GltX, His6-RplK, and SeqA-His6. Plasmid pET28a::gltX was transformed into BL21(DE3) strain. An overnight culture was grown in LB medium containing kanamycin (50 μg/ml), diluted 1000× into 250 ml of LB medium with kanamycin, and grown at 30°C. The expression of gltX was induced at OD600nm of 0.6 with 1 mM IPTG for 2 hours. His6-RplK was produced from the pET28a::rplK plasmid the same way as His6-GltX. SeqA-His6 was produced from the pET28a::seqA plasmid in 750 ml of LB medium the same way as His6-GltX. The expression of seqA was induced at OD600nm of 0.5 with 0.2 mM IPTG for 2 hours.