Western Blot Analysis of Phosphorylation

For Western blot analyses using anti-phosphothreonine (α-pThr), anti-StkP, and anti-PhpP antibodies, bacteria were grown exponentially in 5 mL BHI broth to OD₆₂₀ ≈ 0.1 to 0.2–0.5. Aliquots of 1.0 to 1.5 mL were centrifuged (5 min, 13,000×g at 25 °C), and cell pellets were put on dry ice. Frozen pellets were resuspended in 20 µL SEDS lysis buffer (0.1% deoxycholate (vol/vol), 150 mM NaCl, 0.2% SDS (vol/vol), 15 mM EDTA pH 8.0) for samples with OD₆₂₀ ≈ 0.1–0.2 and 40–80 µL for samples with OD₆₂₀ ≈ 0.4. Samples were incubated at 37°C for 15 min until cells visibly lysed as determined via phase microscopy. The Bio-Rad DC™ protein assay kit I was used to determine total protein concentrations of samples using a standard curve of 0.1 to 3.0 mg/mL of BSA. Absorbance (750 nm) were determined in a 96-well plate reader (Synergy H1 Hybrid Reader, BioTek). Samples were diluted with 2× Laemlli SDS loading buffer (Bio-Rad) and incubated at 95°C for 10 min. 12.5 µg of total protein was loaded per sample onto a 4–15% precast gradient SDS-PAGE gel (Bio-Rad) and subjected to electrophoresis. Proteins were transferred to a nitrocellulose membrane. StkP proteins, PhpP proteins, and threonine-phosphorylated proteins were detected respectively with anti-StkP (1:50,000), anti-PhpP (1:20,000), or α-pThr (1:2000) (Novakova et al., 2010 (link)) as primary antibodies, and ECL anti-rabbit IgG horseradish peroxidase linked whole antibody as secondary (1:10,000). Chemiluminescent signals in protein bands were detected with 1–3 min exposures and quantified using an IVIS imaging system as described in previously (Wayne et al., 2010 (link)). To determine relative amounts of threonine-phosphorylated proteins and the PhpP and StkP proteins, luminescence values for each band were subtracted by the value of the background (defined as a box enclosing the same area of the blot with no detectable signal or from a Δ[phpP-stkP]). Background-subtracted amounts are expressed relative to amounts in wild-type or bacteria that were not depleted for GpsB. After blotting, membranes were stained with amido black solution to confirm that protein loading was similar between samples.