Pro q diamond phosphoprotein blot stain kit

Phosphoproteins were fluorescent stained on PVDF or nitrocellulose membranes using Pro-Q Diamond Phosphoprotein Blot Stain Kit (Thermo Scientific) according to the manufacturer’s instructions. The proteins electroblotted onto PVDF or nitrocellulose membrane were incubated in 7% acetic acid −10% methanol for 10 min, the membrane was washed four times in about 25 mL of dH₂O for about 5 min. In the next step, the membrane was incubated with Pro-Q Diamond phosphoprotein blot stain reagent for 15 min. To remove excess dye from the membrane, the blot was washed three times with 30 mL of 50 mM sodium acetate −20% acetonitrile, pH 4.0. After obtaining results with the Pro-Q^® Diamond phosphoprotein blot stain, the blot was stained with SYPRO^® Ruby protein stain, to ascertain the relative phosphorylation state of proteins. The fluorescent signals were documented using the GelDoc Go Imaging system (www.bio-rad.com) (Bio-Rad).