25. It is strongly advised never to refine the fourth parameter in
this type of analysis, i.e., concentration of the labeled protein.
Its value is too correlated with the other parameters.
26. Most programs show only the square roots of the diagonal
elements of the variance–covariance matrix multiplied by a
factor as the “errors” in the parameters. These are usually
unrealistically low.
27. As emphasized in the upper part of Fig. 9, the T-Jump signal
can be very small. However, it is often very reproducible, as
evidenced by the narrow error bars in the lower part of the
figure.
28. GUSSI is richly featured, and it is extensively documented in its
accompanying manual.
References
1. Jerabek-Willemsen M, Andre ´ T, Wanner R et al
(2014) MicroScale thermophoresis: interaction
analysis and beyond. J Mol Struct
1077:101–113
2. Duhr S, Braun D (2006) Why molecules move
along a temperature gradient. Proc Natl Acad
Sci U S A 103:19678–19682
3. Baaske P, Wienken CJ, Reineck P et al (2010)
Optical thermophoresis for quantifying the
buffer dependence of aptamer binding.
Angew Chem Int Ed 49:2238–2241
4. Wienken CJ, Baaske P, Rothbauer U et al
(2010) Protein-binding assays in biological
liquids using microscale thermophoresis. Nat
Commun 1:100
5. Jerabek-Willemsen M, Wienken CJ, Braun D
et al (2011) Molecular interaction studies
using microscale thermophoresis. Assay Drug
Dev Technol 9:342–353
6. Lippok S, Seidel SAI, Duhr S et al (2012)
Direct detection of antibody concentration
and affinity in human serum using microscale
thermophoresis. Anal Chem 84:3523–3530
7. Seidel SAI, Dijkman PM, Lea WA et al (2013)
Microscale
thermophoresis
quantifies
biomolecular interactions under previously
challenging conditions. Methods 59:301–315
8. Scheuermann TH, Padrick SB, Gardner KH
et al (2016) On the acquisition and analysis of
microscale thermophoresis data. Anal Biochem
496:79–83
9. Tso SC, Chen Q, Vishnivetskiy SA et al (2018)
Using two-site binding models to analyze
microscale thermophoresis data. Anal Biochem
540-541:64–75
10. Brautigam CA (2015) Calculations and
publication-quality illustrations for analytical
ultracentrifugation data. Methods Enzymol
562:109–134
11. Bevington PR, Robinson DK (1992) Data
reduction and error analysis for the physical
sciences. WCB/McGraw-Hill, Boston, MA
12. Pace CN, Vajdos F, Fee L et al (1995) How to
measure and predict the molar absorption coefficient of a protein. Protein Sci 4:2411–2423
13. Fung HYJ, Fu S-C, Brautigam CA et al (2015)
Structural determinants of nuclear export signal orientation in binding to exportin CRM1.
elife 4:e10034
MST of Protein-Ligand Interactions
181
this type of analysis, i.e., concentration of the labeled protein.
Its value is too correlated with the other parameters.
26. Most programs show only the square roots of the diagonal
elements of the variance–covariance matrix multiplied by a
factor as the “errors” in the parameters. These are usually
unrealistically low.
27. As emphasized in the upper part of Fig. 9, the T-Jump signal
can be very small. However, it is often very reproducible, as
evidenced by the narrow error bars in the lower part of the
figure.
28. GUSSI is richly featured, and it is extensively documented in its
accompanying manual.
References
1. Jerabek-Willemsen M, Andre ´ T, Wanner R et al
(2014) MicroScale thermophoresis: interaction
analysis and beyond. J Mol Struct
1077:101–113
2. Duhr S, Braun D (2006) Why molecules move
along a temperature gradient. Proc Natl Acad
Sci U S A 103:19678–19682
3. Baaske P, Wienken CJ, Reineck P et al (2010)
Optical thermophoresis for quantifying the
buffer dependence of aptamer binding.
Angew Chem Int Ed 49:2238–2241
4. Wienken CJ, Baaske P, Rothbauer U et al
(2010) Protein-binding assays in biological
liquids using microscale thermophoresis. Nat
Commun 1:100
5. Jerabek-Willemsen M, Wienken CJ, Braun D
et al (2011) Molecular interaction studies
using microscale thermophoresis. Assay Drug
Dev Technol 9:342–353
6. Lippok S, Seidel SAI, Duhr S et al (2012)
Direct detection of antibody concentration
and affinity in human serum using microscale
thermophoresis. Anal Chem 84:3523–3530
7. Seidel SAI, Dijkman PM, Lea WA et al (2013)
Microscale
thermophoresis
quantifies
biomolecular interactions under previously
challenging conditions. Methods 59:301–315
8. Scheuermann TH, Padrick SB, Gardner KH
et al (2016) On the acquisition and analysis of
microscale thermophoresis data. Anal Biochem
496:79–83
9. Tso SC, Chen Q, Vishnivetskiy SA et al (2018)
Using two-site binding models to analyze
microscale thermophoresis data. Anal Biochem
540-541:64–75
10. Brautigam CA (2015) Calculations and
publication-quality illustrations for analytical
ultracentrifugation data. Methods Enzymol
562:109–134
11. Bevington PR, Robinson DK (1992) Data
reduction and error analysis for the physical
sciences. WCB/McGraw-Hill, Boston, MA
12. Pace CN, Vajdos F, Fee L et al (1995) How to
measure and predict the molar absorption coefficient of a protein. Protein Sci 4:2411–2423
13. Fung HYJ, Fu S-C, Brautigam CA et al (2015)
Structural determinants of nuclear export signal orientation in binding to exportin CRM1.
elife 4:e10034
MST of Protein-Ligand Interactions
181