Pro q diamond

Gels were stained for phosphorylated protein using Pro-Q Diamond (Invitrogen, Carlsbad, CA) according to manufacturer recommendations. Gels were immersed in fixing solution (50% methanol and 10% acetic acid) and incubated twice at room temperature with gentle agitation for 30 min. Fixed gels were then immersed in ultrapure water in order to remove all the methanol and acetic acid. Pro-Q Diamond phosphoprotein gel stain was used to stain the gels for 2 h in the dark, followed by destaining in destaining solution (20% acetonitrile, 50 mM sodium acetate [pH 4]) for 30 min 3 times. Gels were washed with ultrapure water 2 times before they were imaged (532 nm laser; excitation: 555 nm; emission: 580 nm) using a TyphoonTM FLA 9500 biomolecular imager (GE Healthcare). After gel imaging, gels were stained with Sypro Ruby Protein gel stain (Invitrogen, Carlsbad, CA) overnight in the dark and were transferred to a clean container, where they were destained twice with destaining solution (10% methanol, 7% acetic acid) for 30 min and rinsed with ultrapure water. Gels were then imaged (473 nm laser; excitation: 450 nm; emission: 610 nm) utilizing a TyphoonTM FLA 9500 biomolecular imager (GE Healthcare). Gel images stained with Pro-Q Diamond and Sypro Ruby were analyzed using PDQUEST software (Bio-Rad Laboratories Inc.).