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

Abstract

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.

α-synucleinopathy associated calcium overload and autophagy failure is regulated by gain-of-function of Tousled-like kinase

“…40 μmol/L MnCl 2 and 20 μmol/L phos-tag (Wako #304-93521) were added to the SDS-PAGE (8 %). After the electrophoresis, the phos-tag gels were incubating with transfer buffer containing 5 mM EDTA for 20 minutes twice to remove the divalent cations 49 . Washing with transfer buffer for 10 minutes once.…”

Functional and Molecular Characterisation of Heart Failure Progression in Mice and the Role of Myosin Regulatory Light Chains in the Recovery of Cardiac Muscle Function

“…The details of the antibodies used are presented in Table S6 . A comprehensive guide for using Phos-tag gels is provided in [ 104 ]. …”

“…The details of the antibodies used are presented in Table S6. A comprehensive guide for using Phos-tag gels is provided in [104]. The methodology used to quantify enzyme expression was exactly the same as described previously in Section 2.3.…”

Investigating the role of phosphorylated regulatory light chains during heart failure progression

“…The electrophoresis duration, Phos-tag concentration and protein amount to be loaded were systematically optimised to use the gel effectively. The systematic approach for optimisation is recorded in the manuscript published in Current Protocols (273).…”

“…, where 0P, 1P and 2P refers to zero, singly and doubly phosphorylated protein bands, respectively. Although phos-tag gel electrophoresis and western blotting can be a tedious procedure requiring thorough optimisation, it is relatively cheap and produce a reasonable output of data per gel (273). Thus, this was chosen over other techniques such as ELISA and mass spectrometry (273).…”