Development of a homogeneous competitive immunoassay for phosphorylated protein antigen based on the enhanced fluorescence resonance energy transfer technology
Section snippets
Apparatus
Fluorescence spectra were measured using a spectrofluorophotometer RF-5300PC (Shimadzu, Kyoto, Japan) with a quartz crystal cuvette.
Materials
Glutathione-S-transferase (GST)-fused mouse phospho-ERK2 (pERK2) was purchased from Upstate Biotechnology. Anti-diphosphorylated ERK mouse monoclonal antibody, MAPK-YT was purchased from Sigma Aldrich.
Peptide
To prepare molecular probes for competitive enhanced FRET immunoassay, four diphosphorylated peptides corresponding to the active site of ERK, DHTGFL(pT)E(pY)VAT,
Principle of the assay
The principle of competitive enhanced FRET immunoassay for diphosphorylated ERK is shown in Fig. 1. The assay employs two fluorescent conjugates. One is composed of an antibody fragment that binds to the diphosphorylated site of ERK, a flexible linker, and a GFP color variant EYFP with a C-terminally tethered leucine zipper motif. The other is composed of a synthetic diphosphorylated peptide recognized by the antibody fragment, a flexible linker, and another GFP color variant ECFP tethered to
Acknowledgment
We are thankful for the financial support provided by the New Energy and Industrial Technology Development Organization (NEDO) of Japan for this work.
References (20)
- et al.
The p53 tumor suppressor protein, a modulator of cell proliferation
J. Biol. Chem.
(1992) - et al.
A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gel
Anal. Biochem.
(1989) - et al.
Analyzing JNK and p38 mitogen-activated protein kinase activity
Methods Enzymol.
(2001) - et al.
Analysis of mitogen-activated protein kinase activation and interactions with regulators and substrates
Methods
(2006) - et al.
Recent advances in technologies for analyzing protein kinases
J. Pharmacol. Sci.
(2007) Phosphorylation state-specific antibodies: applications in investigative and diagnostic pathology
Am. J. Pathol.
(2003)- et al.
Enhanced fluorescence resonance energy transfer immunoassay with improved sensitivity based on the Fab′-based immunoconjugates
Anal. Biochem.
(2007) - et al.
Electrode-based enzyme immunoassays using urease conjugates
Methods Enzymol.
(1980) - et al.
A heterologous enzyme immunoassay of prostaglandin E2 using a stable enzyme-labeled hapten mimic
Anal. Biochem.
(1988) - et al.
Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes
J. Biol. Chem.
(2006)
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