Application Notes

Competitive assay approach: binding of small molecules to the active form of p38

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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. © 2011 NanoTemper Technologies GmbH

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