Mn2+-Phos-Tag Polyacrylamide for the Quantification of Protein Phosphorylation Levels

• Main Authors: Ferenczi, M.A (Author), Markandran, K. (Author), Shun, L.M (Author), Xuan, J.V.L.E (Author), Yu, H. (Author)
• Format: Article
• Language: English
• Published: John Wiley and Sons Inc 2021
• Subjects: 1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol; acrylic acid resin; Acrylic Resins; Article; electrophoresis; Electrophoresis, Polyacrylamide Gel; human; Humans; image analysis; immunohistochemistry; manganese; mass spectrometry; metabolism; molecular weight; muscle mass; phosphoprotein; Phosphoproteins; phosphoproteomics; phosphorylation; Phosphorylation; Phos-tag; polyacrylamide; polyacrylamide gel electrophoresis; polyvinylidene fluoride; protein expression; protein phosphorylation; pyridine derivative; Pyridines; skeletal muscle; ubiquitination; western blotting; Western blotting
• Online Access: View Fulltext in Publisher
• ISBN: 26911299 (ISSN)
• DOI: 10.1002/cpz1.221

This paper provides a guideline for optimizing and utilizing Mn2+ Phos-tag gel technology to separate phosphorylated proteins from their unphosphorylated counterparts. It provides key insights into methods for careful sample preparation and experimental directions for determining the appropriate Phos-tag gel compositions and electrophoresis and western blotting conditions. This protocol has been used to successfully resolve proteins extracted from cardiac and skeletal muscles. The guidelines can be extended for optimizing protocols to resolve proteins from other cells or tissue sources. With this, phosphoproteomics and the elucidation of underlying mechanisms of disease progression can be accelerated. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.