Purification-free Labeling in Whole Cell Lysate and Binding Characterization by MST

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 Kd 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.