Microscale thermophoresis (MST) is a free-solution method for analyzing protein and small-molecule interactions in biological liquids such as blood serum and cell lysate. This technique uses thermophoresis, the directed motion of molecules in temperature gradients, to determine molecular size, charge, and hydration shell. The study validates MST using systems like human interferon gamma and calmodulin with calcium. It shows that the affinity of quercetin to PKA is 400-fold reduced in serum compared to buffer, highlighting the importance of biological matrix effects. MST is efficient, requiring minimal sample and working in free solution. It is an optical, contact-free method that minimizes contamination. The technique uses a fluorescent label on one binding partner to visualize thermophoretic changes. The method allows quantification of dissociation constants in both buffer and biological environments, such as blood serum or cell lysate. The study demonstrates MST's ability to analyze protein-protein interactions, low-molecular-weight binders, and ion binding. For example, MST quantifies the binding of CaM to Ca²+ ions and the interaction of quercetin with PKA. It also shows that MST can detect binding in complex biological matrices, such as cell lysate and serum, where affinities are significantly reduced due to factors like competitive interactions or changes in viscosity. MST is suitable for studying interactions in biological liquids, providing insights into protein functionality and aiding drug development. It is particularly useful for analyzing low-affinity interactions and small molecules, which are often challenging with other techniques. The method is fast, requires minimal sample, and can be applied to a wide range of biological samples, including crude cell extracts and serum. The study also highlights the importance of considering biological matrices when interpreting binding data, as they can significantly affect apparent affinities. MST provides a reliable alternative to traditional methods like surface plasmon resonance, offering high sensitivity and the ability to quantify interactions in complex environments. The technique is valuable for studying biomolecule interactions in biology and drug development, as it can detect even minor changes in binding behavior.Microscale thermophoresis (MST) is a free-solution method for analyzing protein and small-molecule interactions in biological liquids such as blood serum and cell lysate. This technique uses thermophoresis, the directed motion of molecules in temperature gradients, to determine molecular size, charge, and hydration shell. The study validates MST using systems like human interferon gamma and calmodulin with calcium. It shows that the affinity of quercetin to PKA is 400-fold reduced in serum compared to buffer, highlighting the importance of biological matrix effects. MST is efficient, requiring minimal sample and working in free solution. It is an optical, contact-free method that minimizes contamination. The technique uses a fluorescent label on one binding partner to visualize thermophoretic changes. The method allows quantification of dissociation constants in both buffer and biological environments, such as blood serum or cell lysate. The study demonstrates MST's ability to analyze protein-protein interactions, low-molecular-weight binders, and ion binding. For example, MST quantifies the binding of CaM to Ca²+ ions and the interaction of quercetin with PKA. It also shows that MST can detect binding in complex biological matrices, such as cell lysate and serum, where affinities are significantly reduced due to factors like competitive interactions or changes in viscosity. MST is suitable for studying interactions in biological liquids, providing insights into protein functionality and aiding drug development. It is particularly useful for analyzing low-affinity interactions and small molecules, which are often challenging with other techniques. The method is fast, requires minimal sample, and can be applied to a wide range of biological samples, including crude cell extracts and serum. The study also highlights the importance of considering biological matrices when interpreting binding data, as they can significantly affect apparent affinities. MST provides a reliable alternative to traditional methods like surface plasmon resonance, offering high sensitivity and the ability to quantify interactions in complex environments. The technique is valuable for studying biomolecule interactions in biology and drug development, as it can detect even minor changes in binding behavior.