We measured the capacity of the c-Abl–SH2 domain to bind to synthetic peptides using ELISA. Briefly, synthetic peptides (50 μg/ml) (biotin-GGA-STYPKSPDSRQPEPLY [*p] ENVNVVSGNEVYSLV and biotin-GGA-STYPKSPDSRQPEPLYENVNVVSGNEVYSLV) were diluted fourfold in PBS buffer to create a concentration gradient and coated onto Nunc MaxiSorp ELISA plates overnight at 4°C. The plates were further blocked with 0.3% (w/v) gelatin in PBS buffer for 2 hours at 37°C, followed by the addition of GST (10 μg/ml) or GST-SH2 fusion proteins for 1 hour at 37°C. The plates were then washed with PBST buffer (PBS with 0.05% Tween 20) and incubated with mouse anti-GST antibody (clone N100/13, NeuroMab) for 1 hour at room temperature. After washing, plates were incubated with horseradish peroxidase (HRP)–conjugated goat anti-mouse Ig (00090941, Dako) as the second antibody for 45 min at room temperature. After washing, the substrate solution, comprising 0.325% o-phenylenediamine dihydrochloride (Sigma-Aldrich) and 0.085% H2O2 in 0.3 M tris-citrate buffer (pH 6.0), was added. After incubation at room temperature for 10 min in the dark, the reaction was stopped with 2.5 M H2SO4, and the plates were measured using an ELISA plate reader (Bio-Rad) for light absorbance at 490 nm.

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