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Although Phos-tag SDS-PAGE is widely used to resolve phosphorylated protein isoforms, its reproducibility and quantitative reliability are often limited by variability in the key experimental factors, including gel composition, electrophoretic conditions, protein loading, and sample preparation. Here, we present a standardized Mn\u00b2\u207a\u2013Phos-tag SDS-PAGE workflow optimized for cardiac myofilament proteins, using myosin regulatory light chain 2 (MLC2) and cardiac troponin I (cTnI) as model targets. We systematically evaluated critical parameters\u2014including Mn\u00b2\u207a and Phos-tag concentrations, acrylamide composition, electrophoretic regime, buffer chemistry, protein loading, and EDTA-mediated transfer\u2014to define conditions that maximize phospho-species resolution while preserving quantitative fidelity. We further demonstrate that electrophoresis rate, sample loading, and extraction strategy significantly influence band morphology, signal intensity, and the apparent distribution of phospho-species. As a use case scenario, we compared Trichloroacetic acid (TCA)-extracted mouse left ventricular homogenates with myofibrils prepared using a widely adopted Triton-X-100 tissue-demembranization protocol. Myofibril preparation was associated with profound MLC2 dephosphorylation at the earliest stages of preparation, whereas cTnI exhibited a marked reduction in higher-order, low-stoichiometry phosphoforms. Further evaluation of Myosin-binding protein C (MyBP-C) showed progressive loss of phosphorylation over the course of 24 hours. We submit that TCA-extracted heart standards in combination with Phos-tag gels can provide valuable quality control for the phosphorylation status of myofibril preparations, and that inclusion of a high-affinity PP2A and PP1 phosphatase inhibitor like okadaic acid may benefit future myofibril mechanics studies.<\/jats:p>","DOI":"10.64898\/2026.08.21.746362","type":"posted-content","created":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T14:35:18Z","timestamp":1787582118000},"source":"Crossref","is-referenced-by-count":0,"title":["Optimized Mn\u00b2\u207a\u2013Phos-tag Gels Reveal Sarcomeric Protein Dephosphorylation upon Myofibril Preparation"],"prefix":"10.64898","author":[{"given":"Sunayana Begum","family":"Syed","sequence":"first","affiliation":[{"name":"Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Axel","family":"Fenwick","sequence":"additional","affiliation":[{"name":"Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Skylar M. L.","family":"Bodt","sequence":"additional","affiliation":[{"name":"Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rohan","family":"Wishard","sequence":"additional","affiliation":[{"name":"Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"D. 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