Pro q diamond solution

Mouse hearts were minced into small pieces with scissors and homogenized in F60 solution (60 mM KCl, 30 mM imidazole, 2 mM MgCl₂, and a protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific, 78440)). Tissues were collected by centrifugation for 3 minutes at 8,000 x g, resuspended in F60 solution two more times, followed by 2 washes with F60 solution containing 1% Triton x-100. The pellet was washed 3 times with F60 and eluted with buffer (20 mM HEPES, 1% Triton, 0.5 M NaCl, 1 mM EDTA, and a protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific, 78440)). Protein samples were quantified and equal amounts of protein was subjected to 12% SDS-PAGE. For Pro-Q Diamond staining, the acrylamide gel containing the separated proteins were fixed, washed and stained with Pro-Q Diamond solution (Invitrogen, P-33300). Images were visualized through UV transillumination (Bio-Rad). For Phos-Tag gels, Phos-Tag (Wako Chemicals, 304-93526) and MnCl₂ solution were added to the 12% acrylamide gel to reach a final concentration of 50 µM, and 0.1 mM respectively. The gel was washed with transfer buffer containing 1 mM EDTA, transferred, and blotted with MLC2V antibody (Proteintech, 10906-1-AP).