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Affinity Chromatography of Phosphorylated Proteins
Grigoriy S. Tchaga
Summary
This chapter covers the use of immobilized metal ion affinity chromatography (IMAC)
for enrichment of phosphorylated proteins. Some requirements for successful enrichment
of these types of proteins are discussed. An experimental protocol and a set of application
data are included to enable the scientist to obtain high-yield results in a very short time
with pre-packed phospho-specific metal ion affinity resin (PMAC).
Key Words: Phosphorylated proteins; immobilized metal ion affinity chromatography;
ferric protein purification.
1. Introduction
Protein phosphorylation is a highly important mechanism for signal transduction
in eukaryotic cells, and there are examples of phosphorylation events
occurring in prokaryotic organisms as well (1–5).
Signal transduction, transcriptional regulation, and cell division are just three
examples of the many metabolic processes regulated by the phosphorylation and
dephosphorylation of proteins by kinases and phosphatases. Despite the broad
use of phosphorylation to regulate cellular processes, only a small percentage
of all cellular proteins are phosphorylated at any given time (6–7).
The target proteins are prevalently phosphorylated on side chains that contain
a hydroxyl group, such as serine, threonine, and tyrosine residues. However,
an increasing number of examples of histidine phosphorylation have also been
described (4). Abundance of the four different phosphorylated side chains in
From: Methods in Molecular Biology, vol. 421: Affinity Chromatography: Methods and Protocols, Second Edition
Edited by: M. Zachariou © Humana Press, Totowa, NJ
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