Pro q diamond phosphoprotein gel destaining solution

His-tagged (1 μg) PKA Cα1 (Millipore, Billerica, MA, USA,) or his-tagged (1 μg) CK2 (ATGen Ltd, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea,) was incubated with purified AMBN-WT (100 pmol), C-terminus (314 pmol), N-terminus (100 pmol), or DelEx5 (100 pmol) in CK2 or PKA reaction buffer supplied with 1 mM ATP in a total volume of 20 μl at 30°C for 24 h. Ten picomoles from each of the samples were submitted to LC-ESI-MS analyses. The remaining of the samples were boiled in Laemmli buffer, loaded on a 12% Ready Gel Tris-HCL (BioRad), and separated by electrophoresis, and the gel stained for phospho-proteins with Pro-Q® Diamond Phosphoprotein Gel Stain and destained with Pro-Q® Diamond Phosphoprotein Gel Destaining Solution (ThermoFisher Scientific, Waltham, MA, USA) according to manufacturer's protocol. The phosphor luminiscent gel bands were then scanned at in ChemiDoc XRS+ imaging system (BioRad) at 510 nm.

The level of titin phosphorylation was determined using a previously described method^{65 (link)} with minor modifications. The native level of protein phosphorylation was estimated in the gels using a Pro-Q Diamond fluorescent dye (ThermoFisher Scientific). The gels were incubated in an aqueous solution of 50% EtOH and 10% acetic acid for 12–18 h, washed with distilled water for 30 min and stained for 1.5 h. The gels were then washed with Pro-Q Diamond phosphoprotein gel destaining solution (ThermoFisher Scientific), and protein bands containing phosphate groups were visualised using a Bio-Rad ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Finally, the gels were stained with Coomassie Brilliant Blue G-250 and R-250 mixed at a 1:1 ratio to estimate the total protein content.