Phosphoprotein Detection and Visualization

Staining of phosphorylated proteins was performed using Pro-Q™ Diamond Phosphoprotein Gel Stain (Invitrogen) following protein separation on SDS-PAGE. The indicated proteins were incubated for 30 min at 30 °C in the kinase buffer containing 25 mM Tris-Cl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 1 mM PMSF, 25 μM ATP. After separation on 10% SDS-PAGE, the gel was fixed with 50% methanol and 10% acetic acid overnight. The gel was washed with water for 30 min and stained with 3x diluted Pro-Q diamond stain (Invitrogen) in the dark for 2 h. The gel was destained four times for 30 min each with 20% acetonitrile, 50 mM sodium-acetate (pH 4.2). The gel was washed again with water for 10 min and was scanned at 400 V using a Typhoon Scanner (GE Healthcare)^{51 (link)}.