Authors: Junya Tomida , Hiroyuki Kitao , Eiji Kinoshita & Minoru Takata
Detection of phosphorylation by western blotting is an important procedure to elucidate molecular mechanisms in signal transduction pathways involving kinases and phosphatases. Anti-phospho-tyrosine monoclonal antibodies have been widely used because they react with plethora of proteins containing phosphorylated tyrosine residues. In contrast, the monoclonal antibodies against phospho-serine or threonine residues are unpopular, since their affinity and specificity are less than optimal. To achieve precise characterization of signaling events, it is desirable to raise a good anti-phospho-site-specific antibody to clearly detect phosphorylated species. However, raising this type of antibody is costly and time-consuming, and sometimes results in failure.
Use of Phos-tag may provide an alternative method to detect phosphorylated proteins. Phos-tag is a dinuclear metal complex that acts as a novel phosphate-binding tag. Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. Phosphorylated proteins can be detected as slower migrating species by electrophoresis and western blotting using PAGE gel containing appropriate amount of Phos-tag acrylamide.
Previously Phos-tag acrylamide has been used in 20-100 μM concentrations. We have now found that lower concentrations (3.5-5 μM) of Phos-tag can dramatically improve the separation between phosphorylated and non-phosphorylated species of a protein with larger molecular mass (~150 kDa). Here we describe a detailed protocol that includes tips to ensure easier application of this methodology. The procedure should be carried out as in the standard SDS-PAGE unless stated otherwise.
Transfer buffer (Tris 25 mM/ Glycine 0.192M/ 20% methanol)
Other reagents including sample loading buffer and gel running buffer are the same as the standard SDS-PAGE method.
Preparation of SDS-PAGE gel containing Phos-tag
Running the gel
Blocking and detection
Figure 1: Phosphporylation and monoubiuqitination of chicken FANCI.
DT40 cells were treated with MMC 500ng/ml for 6h or left untreated. Whole cell lysate were prepared and blotted with anti-GFP. Wild type DT40 (WT), fanci cells expressing GFP-chicken FANCI lacking monoubiuqitination site (K563R) or carrying alanine substitutions in six putative phosphorylation sites (Ax6) were analyzed. In this condition (Phos-tag 25 μM or 50 μM), it is difficult to clearly separate phosphorylated and non-phosphorylated FANCI.
Figure 2: Improved Phos-tag western blotting.
Lysates were similarly prepared as in Figure 1. Electrophoresis and western blotting were done as described in this protocol.
FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway, Masamichi Ishiai, Hiroyuki Kitao, Agata Smogorzewska, Junya Tomida, Aiko Kinomura, Emi Uchida, Alihossein Saberi, Eiji Kinoshita, Emiko Kinoshita-Kikuta, Tohru Koike, Satoshi Tashiro, Stephen J Elledge, and Minoru Takata, Nature Structural & Molecular Biology 15 (11) 1138 - 1146 19/10/2008 doi:10.1038/nsmb.1504
Junya Tomida & Minoru Takata, Laboratory of DNA Damage Signaling, Radiation Biology Center, Kyoto University
Hiroyuki Kitao, Department of Molecular Oncology, Graduate School of Medical Sciences, Kyushu University
Eiji Kinoshita, Dept of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University
Source: Protocol Exchange (2008) doi:10.1038/nprot.2008.232. Originally published online 20 October 2008.