Pro q diamond phosphoprotein gel distaining solution

To detect overall phosphorylation changes, a 15% SDS-PAGE gel was stained with Pro-Q Diamond Phosphoprotein Gel Stain (Invitrogen, P33300, Carlsbad, CA) and destained with Pro-Q Diamond Phosphoprotein Gel Distaining Solution (Invitrogen, P33310, Carlsbad, CA). The gel was then stained with Coomassie G-250 Stain (Bio-Rad Inc., 1,610,786, Hercules, CA) to determine total protein levels. Gel images were captured with ChemiDoc MP (Bio-Rad, Inc., 13,036, Hercules, CA) and band densities were determined and analyzed by using ImageLab 6.0.1 software and Microsoft Excel.

Determination of titin phosphorylation level was carried out by the method described in [36 (link)] with negligible modifications. A native level of protein phosphorylation in gel was estimated with the help of fluorescent stain Pro-Q Diamond (Invitrogen) for phosphoproteins. For this purpose, gels were put into the solution containing 50% of ethanol and 10% of acetic acid for 12–18 hours and, after 30 min washing in distilled water, were stained for 1.5 hours. The stained gel was washed off in a Pro-Q Diamond phosphoprotein gel distaining solution (Invitrogen). Protein bands containing phosphate were viewed using Bio-Rad system Pharos. Then gels were stained with Coomassie Brilliant Blue G-250 and R-250 mixed in a 1 : 1 ratio for the control estimation of protein content.