Figure 4

Comparison of SW620 cytosolic proteins binding to LckSH2 versus LckSH2 pre-blocked with a high affinity binding pY peptide. Cytosol (S100) of SW620 cells was pre-cleared several times with GST immobilised on GSH beads to reduce non-specific protein binding. Supernatants of these pre-clearings corresponding to 20 mg of S100 were then incubated with bead-immobilised GST-LckSH2 or GST-LckSH2 pre-blocked with the peptide EPQpYEEIPI, or unloaded GSH beads to detect potentially remaining non-specific protein binding and, after washing repeatedly with RIPA buffer, separated by SDS-PAGE and stained with colloidal dye (Roti-Blue). The GST-LckSH2 precipitation lane was subsequently processed for MS identification of bound proteins.