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A semi-synthetic approach for tagging and identifying novel
kinase substrates utilizing a thiophosphate ester specific
monoclonal antibody, clone 51-8
Sung M. Lee and Abe Couse
Epitomics, Inc., 863 Mitten Road, Suite 103, Burlingame, CA, Email: info@epitomics.com, Web: www.epitomics.com
Introduction
Protein phosphorlyation is the most widespread and studied
regulatory signaling mechanism occurring in both eukaryotic
and prokaryotic organisms. The mechanism of adding
phosphate (PO4) to a protein by a kinase forms the basis of
cell signaling networks involved in all fundamental cellular
processes (1–3). However, the biochemical identification
of the substrates for any kinase of interest (KOI) has been
challenging and limited. This is due to the low abundance
of most phosphoproteins, coupled with substoichiometric
phosphorylation, and weak affinity of kinase-substrate interactions. Here we describe and illustrate the semi-synthetic
kinase substrate tagging and detection technique using a
novel thiophosphate ester specific monoclonal antibody first
introduced by Allen et al. (5,6).
the thiophosphate ester specific monoclonal antibody (clone
51-8; Epitomics, Inc. Burlingame, CA, USA) at 1:5000.
Antibodies were incubated overnight at 4°C. α-GST antibody
confirmed equal loading (Figure 2).
Conclusion
By utilizing the novel rabbit monoclonal antibody (clone
51-8) to detect proteins containing thiophosphate ester
site following labeling, one can identify both known and
novel substrates as well as detect context independent
serine, threonine, and tyrosine phosphorylated substrates.
This semi-synthetic epitope tagging provides a new way
to identify and isolate substrates of various kinases (4–6).
Visit www.epitomics.com/kinase to learn more.
References
Methods
The semi-synthetic epitope tagging reaction was carried out
as in Figure 1. First a KOI (JNK1, 10 ng) was incubated with
c-jun-GST substrate (1 µg), with ATPγS as a thiophosphate
donor. Thiophosphorylated c-jun-GST was then alkylated with
p-nitrobenzyl mesylate (PNBM, 2.5 mM). The kinase reaction
was carried out for 30 min at room temperature. Then PNBM
alkylation (2.5 mM) proceeded for 2 h at room temperature.
Reaction products were then analyzed by Western blot using
1. Johnson, S.A. and Hunter, T. Nature Methods 2, 17–25, 2005.
2. Hunter, T. Signaling−2000 and beyond. Cell 100, 113−127, 2000.
3. Cozzon, A.J. Ann. Rev. Microbiol. 42, 97–125, 1988.
4. Cai, D, Article Review, Mol. BioSyst., 3, 516 – 517, 2007.
5. Allen, J.J. et al. J. Am. Chem. Soc., 127(15), 5288–5289, 2005.
6. Allen, J.J. et al. Nature Methods, 4(6), 511–516, 2007.
Sponsored Paper. BioTechniques 47:880 (October 2009)
doi 10.2144/000113273
Figure 2. Western blot analysis of semisynthetic tagged kinase substrate using
clone 51-8.
Figure 1. Reaction sequence for affinity tagging kinase substrates.
Vol. 47 | No. 4 | 2009
880
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