TY - JOUR
T1 - Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions
AU - Seidel, Susanne A.I.
AU - Dijkman, Patricia M.
AU - Lea, Wendy A.
AU - van den Bogaart, Geert
AU - Jerabek-Willemsen, Moran
AU - Lazic, Ana
AU - Joseph, Jeremiah S.
AU - Srinivasan, Prakash
AU - Baaske, Philipp
AU - Simeonov, Anton
AU - Katritch, Ilia
AU - Melo, Fernando A.
AU - Ladbury, John E.
AU - Schreiber, Gideon
AU - Watts, Anthony
AU - Braun, Dieter
AU - Duhr, Stefan
N1 - Funding Information:
The authors thank Dr. Alan Goddard and Eleanor Healey for making AlexaFluor488-neurotensin. We also thank Dr. Masoud Vedadi (University of Toronto) for provision of the G9a protein. Dr. Mohammed Yousef (Bio-Rad Laboratories) is thanked for his expert advice during the initial chip set up on the ProteOn system. In addition Peter Röttgermann and Christof Mast are thanked for help with figures. The work of S.A.I.S. and D.B. was supported by the Nano Initiative Munich (NIM) , the Center for NanoScience (CeNS) , the Ludwig Maximilians Universität München (LMU) Initiative Functional Nanosystems and the Deutsche Forschungsgemeinschaft. The work by J.S.J. and I.K. was supported by the NIH Common Fund’s Structural Biology Program Grant P50 GM073197 . The work of P.M.D. and A.W. was supported by the Medical Research Council (MRC) grant G0900076 to A.W. The work by P.S., W.A.L., and A.S. was supported in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases and the National Center for Advancing Translational Sciences, US National Institutes of Health.
PY - 2013/3/1
Y1 - 2013/3/1
N2 - Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science.
AB - Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science.
KW - Binding affinity
KW - Bioliquids
KW - Cooperativity
KW - Dimerization
KW - Label-free
KW - Microscale thermophoresis
UR - http://www.scopus.com/inward/record.url?scp=84884497350&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84884497350&partnerID=8YFLogxK
U2 - 10.1016/j.ymeth.2012.12.005
DO - 10.1016/j.ymeth.2012.12.005
M3 - Review article
C2 - 23270813
AN - SCOPUS:84884497350
SN - 1046-2023
VL - 59
SP - 301
EP - 315
JO - Methods
JF - Methods
IS - 3
ER -