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Mn 2+ ‐Phos‐Tag Polyacrylamide for the Quantification of Protein Phosphorylation Levels
Author(s) -
Markandran Kasturi,
Xuan Jane Vanetta Lee En,
Yu Haiyang,
Shun Lim Meng,
Ferenczi Michael A.
Publication year - 2021
Publication title -
current protocols
Language(s) - English
Resource type - Journals
ISSN - 2691-1299
DOI - 10.1002/cpz1.221
Subject(s) - phosphoproteomics , phosphorylation , blot , polyacrylamide gel electrophoresis , phos , chemistry , chromatography , biochemistry , protein phosphorylation , computational biology , microbiology and biotechnology , biology , protein kinase a , enzyme , gene
This paper provides a guideline for optimizing and utilizing Mn 2+ 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.