Application Notes

Purification-free labeling in whole cell lysate and binding characterization by MST

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APPLICATION NOTE Protein Labeling Purification-free Labeling in Whole Cell Lysate and Binding Characterization by MST Christian Kleusch 1 , Thomas Vercruysse 2 , Dennis Breitsprecher 1 , Dirk Daelemans 2 , Katarzyna Walkiewicz 1 1 NanoTemper Technologies GmbH, Floessergasse 4, 81369 Munich 2 Rega Institute for Medical Research, Virology & Chemotherapy, Minderbroederstraat 10, B-3000 Leuven Abstract For researchers performing biophysical analysis of proteins, a common hurdle encountered is having sufficient amounts of materials with the appropriate purity to perform detailed analysis. Here we demonstrate the utility of a RED-tris-NTA dye from NanoTemper Technologies that can be used to specifically label His- tagged proteins for MicroScale Thermophoresis (MST) binding studies directly in cell lysates. The procedure requires minute amounts of sample, can be carried out without additional lab equipment and accurate K d measurements are obtained within 45 minutes from cell lysis to measurement. As an example, we measured the affinity of a small molecule inhibitor to His-tagged p38α kinase expressed in mammalian cells. Our data demonstrate that MST assays are a rapid and cost- effective method for determination of affinities using unpurified proteins, and thus serves as a powerful tool in the early drug discovery, especially for proteins that are difficult to purify. Introduction Detailed biophysical characterization, whether for basic research or early drug discovery analysis, requires starting material of the highest quality and purity to generate meaningful results. Therefore, suitable purification protocols must be established to produce proteins at high enough concentration and quality. Also, biophysical measurements are typically carried out in artificial buffer systems, which can dramatically affect the outcome of binding studies when compared to the in vivo situation where natural ligands, substrates and ions are present. This study shows

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