Umax powerlook 1120

DesR autophosphorylation reactions were performed with protein concentrations ranging from 0.2 to 500 µM at 25°C and pH 8 and incubation with a mixture of 50 mM acetyl phosphate, 300 mM NaCl, 50 mM MgCl₂, and 20 mM Tris-HCl. The reaction volumes varied between 20 and 100 µl. Addition of acetyl phosphate was immediately followed by warming from 4 to 25°C. Autophosphorylation was stopped at 1 h, the mixture was cooled to 4°C, and SDS-PAGE sample buffer with 2.5 mM DTT was added. Protein samples (0.13 to 1.1 µM) were incubated with SDS-PAGE sample buffer at room temperature for 5 min and mixed with iodoacetamide (40 mM) for 10 min. Phos-tag SDS-PAGE was performed as previously described (57 (link)), with 50 µM Phos-tag acrylamide and 100 µM ZnCl₂. Coomassie-stained gels were scanned with UMAX PowerLook 1120 and LabScan 5.0 (GE HealthCare) and analyzed by Image Quant TL v2005 software (GE HealthCare). The peak areas corresponding to phosphorylated (slower migration) and unphosphorylated species were integrated, and the degree of phosphorylation (ξ_P) was calculated according to the formula ξ_P = DesR − P peak area × (DesR − P peak area + DesR peak area)⁻¹.