Pan anti propionyl lysine antibody

The strains and plasmids used in this study were listed in Table 2. Bacillus subtilis 168 and E. coli were grown on Luria-Bertani (LB) medium. When needed, antibiotics were added to the medium at the following concentrations: ampicillin, 100 μg/ml; kanamycin, 50 μg/ml. All media were sterilized by autoclaving at 121 °C for 20 min.Table 2

The strains and plasmids used in this study.

Strain or plasmid	Source or reference
strains
Bacillus subtilis 168	ATCC 6051
E. coli DH5α	TransGen Biotech
E. coli BL21(DE3)	TransGen Biotech
E. coli BL21(DE3)-BsAcsA	In this work
E. coli BL21(DE3)-BsAcuA	In this work
Salmonella enterica Δacs (JE7758)	18 (link)
plasmids
pET-28a	Thermo Scientific
pBAD30	18 (link)
pGEX-4T-2	Thermo Scientific
pET-BsAcsA	Lab stock
pGEX-BsAcuA	In this work

The antibodies and columns for purification of proteins used were as follows: anti-acetyl-lysine antibody (ImmuneChem Pharmaceuticals, Burnaby, CA); pan anti-propionyl-lysine antibody (PTM Biolabs); HRP conjugates (anti-PrK; ImmuneChem); glutathione transferase (GST) agarose column and nickel-nitrilotriacetic acid (Ni-NTA) superflow column (Qiangen, Valencia, CA).

Protein (10 μg) was isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose filter membrane and maintained at 100 V for 90 min. Subsequently, the membrane was blocked by the use of B-PBST buffer (5% bovine serum albumin [BSA]–PBST buffer [PBS buffer with 0.1% Tween 20]) for 60 min. The membranes were then incubated with pan-antiacetyllysine antibody (PTM Biolabs, Hangzhou, China), pan-antipropionyllysine antibody (PTM Biolabs, Hangzhou, China), pan-antiphosphoserine antibody (ImmuneChem, Burnaby, Canada), or pan-antiphosphothreonine antibody (ImmuneChem, Burnaby, Canada) at 4°C overnight. The membranes were washed with PBST three times and then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:5,000)–PBST at room temperature for 1 h. Next, the membranes were washed with PBST three times and then treated with chemiluminescent HRP substrate (Millipore, Temecula, CA). After that, the samples were used for signal detection.