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