3
analysis. Furthermore this approach also can be
used to screen for compounds that interrupt
protein-protein, protein-peptide or protein-nucleic
acid interactions.
Material and Methods
Assay buffer
For the experiment described here, a 50 mM Tris
buffer pH 7.6 containing 150 mM NaCl, 10 mM
MgCl
2
and 0.05 % Tween-20 has been used.
Instrumentation
The measurements were done on a Nanotemper
Monolith NT.115 instrument.
For evaluation of the tracer199 (Invitrogen)
binding to p38 10 μl of a 50 nM stock solution of
tracer199 were mixed with 10 μl of a serial dilution
of p38 starting at 1 μM. The samples were
incubated for 10 minutes and measured at an
MST power of 15 % and a LED power of 50 %
with a laser-on time of 30 seconds and a laser-off
time of 5 seconds.
For the competition experiment a stock solution of
150 nM p38 and 25 nM tracer199 was prepared.
In this concentration range a sufficient amount of
the fluorescent tracer is bound to the protein.
Please note that not all tracer molecules have to
be in complex with the protein. However, free
tracer will decrease the amplitude of the MST
signal. 10 μl of the stock solution of the tracer199-
p38 complex was mixed with a serial dilution of
the small molecule SB203580 starting at 4 μM and
measured under the same conditions as before.
References
Dominuez et al., p38 MAP kinase inhibitors: many are made,
but few are chosen.
Curr Opin Drug Discov Devel. 2005 Jul;8(4):421-30.
Davis et al.,
Specificity and mechanism of action of some commonly used
protein kinase inhibitors.
Biochem J. 2000 Oct 1;351(Pt 1):95-105.
Lee and Dominguez, MAP kinase p38 inhibitors: clinical
results and an intimate look at their interactions with p38alpha
protein.
Curr Med Chem. 2005;12(25):2979-94.
Pargellis et al., Inhibition of p38 MAP kinase by utilizing a
novel allosteric binding site.
Nat Struct Biol. 2002 Apr;9(4):268-72.
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