interaction. The experimenter may need to adjust the dye
concentration to achieve the desired labeling efficiency.
9. For this protocol, we used a cuvette that holds 150 μL and did
not recover the measured protein solution.
10. The goal of labeling is to achieve sub-stoichiometric labeling
(i.e., ϕ < 1.0). Greater labeling efficiencies increase the likelihood that multiple lysines on the protein’s surface have been
labeled, which can hamper the interaction.
11. The optimal concentration of the protein is well below K D ,
e.g., 0.1K D or lower. However, this is difficult to know a
priori. We have designed this experiment with a final α-CT of
50 nM; in this case, K D ~ α-CT. Although this is not ideal,
Monte Carlo simulations of typical MST data demonstrate that
accurate parameters can still be attained under this
circumstance [8].
12. Do not use a cell phone within 5 m of the instrument. The
electronic noise can cause artifacts in the fluorescence traces.
13. Aberrations in post-run capillary scans indicate that the protein
is likely unstable at the higher temperature induced by the IR
laser. Lower MST powers or adding stabilizing agents (e.g.,
glycerol) to the buffer should be explored.
14. Very high LED powers, i.e., >75%, are best avoided, as they
may result in significant photobleaching of the fluorophore.
15. Protein sticking to the capillaries can be addressed by adding
detergent (as in the Tween already included in this study) or a
“carrier protein” to passivate sticky surfaces. Many researchers
consider bovine serum albumin (BSA) to be an excellent carrier
protein, but we have observed artifacts when using it [8]. If
studying interactions other than the one presented hereinabove, we have found SBTI to have excellent properties for
this purpose.
16. PALMIST’s methods are documented in the literature [8, 9],
and the software comes with a detailed manual describing its
usage.
17. High-speed centrifugation, addition of detergents, and additional purification are candidate treatments for aggregation.
18. If no signal is observed, it is advisable to try other settings, e.g.,
“T-Jump,” which compares fluorescence before the IR laser
actuation to that just after. Also, it is possible to try higher MST
powers or labeling with other dyes. If studying two proteins,
the other binding partner could alternatively be labeled.
19. It is imperative that the buffer composition of the entire dilution series must be the same; i.e., the buffer containing the
MST of Protein-Ligand Interactions
179
concentration to achieve the desired labeling efficiency.
9. For this protocol, we used a cuvette that holds 150 μL and did
not recover the measured protein solution.
10. The goal of labeling is to achieve sub-stoichiometric labeling
(i.e., ϕ < 1.0). Greater labeling efficiencies increase the likelihood that multiple lysines on the protein’s surface have been
labeled, which can hamper the interaction.
11. The optimal concentration of the protein is well below K D ,
e.g., 0.1K D or lower. However, this is difficult to know a
priori. We have designed this experiment with a final α-CT of
50 nM; in this case, K D ~ α-CT. Although this is not ideal,
Monte Carlo simulations of typical MST data demonstrate that
accurate parameters can still be attained under this
circumstance [8].
12. Do not use a cell phone within 5 m of the instrument. The
electronic noise can cause artifacts in the fluorescence traces.
13. Aberrations in post-run capillary scans indicate that the protein
is likely unstable at the higher temperature induced by the IR
laser. Lower MST powers or adding stabilizing agents (e.g.,
glycerol) to the buffer should be explored.
14. Very high LED powers, i.e., >75%, are best avoided, as they
may result in significant photobleaching of the fluorophore.
15. Protein sticking to the capillaries can be addressed by adding
detergent (as in the Tween already included in this study) or a
“carrier protein” to passivate sticky surfaces. Many researchers
consider bovine serum albumin (BSA) to be an excellent carrier
protein, but we have observed artifacts when using it [8]. If
studying interactions other than the one presented hereinabove, we have found SBTI to have excellent properties for
this purpose.
16. PALMIST’s methods are documented in the literature [8, 9],
and the software comes with a detailed manual describing its
usage.
17. High-speed centrifugation, addition of detergents, and additional purification are candidate treatments for aggregation.
18. If no signal is observed, it is advisable to try other settings, e.g.,
“T-Jump,” which compares fluorescence before the IR laser
actuation to that just after. Also, it is possible to try higher MST
powers or labeling with other dyes. If studying two proteins,
the other binding partner could alternatively be labeled.
19. It is imperative that the buffer composition of the entire dilution series must be the same; i.e., the buffer containing the
MST of Protein-Ligand Interactions
179