3
Material and Methods
Assay conditions
Binding of labeled anti-toxin antibody to
SYD977 and SYD985 in PBST buffer:
Protein Labeling: 7 µM murine IgG2a anti-toxin
antibody was labeled in PBS buffer using the
Protein Labeling Kit RED-NHS (L001,
NanoTemper Technologies, Germany).
Sample Preparation: The concentration of the
labeled anti-toxin antibody was kept constant at
1.5 nM. For the interaction analysis of SYD985 and
SYD977 with the labeled anti-toxin antibody, SYD
977 or SYD985 was titrated in 1:1 dilutions. The
highest concentration of SYD977 or SYD985 was
800 nM. All dilutions were performed in PBST-
buffer (PBS + 0.05 % Tween-20). The samples
were spun down 5 min at 4 °C, and then filled into
MST Premium Coated capillaries (Cat# K005,
NanoTemper Technologies, Germany).
Binding of labeled anti-toxin antibody to
SYD985 in human plasma containing buffer:
SYD985 was titrated in 1:1 dilutions in PBST.
Then, NT647-labeled anti-toxin antibody was
added into 500 µl human plasma to a final
concentration of 3 nM. 10 µl of NT647-labeled anti-
toxin-containing human plasma was mixed with
10 µl SYD985 dilutions. The samples were spun
down 5 min at 4 °C, and then filled into MST
Premium Coated capillaries (Cat# K005,
NanoTemper Technologies, Germany).
Instrumentation: The measurements were
conducted on a NanoTemper Monolith NT.115
instrument, at 10 % LED power and 80 % MST
power.
References
1. Chari, R.V.J., Miller, M.L., Widdison, W.C.
. (2014) Antibody–Drug Conjugates: An
Emerging Concept in Cancer Therapy.
Angew. Chem. Int. Ed. 2014, 53, 3796 –
3827
2. Jerabek-Willemsen, M., André, T.,
Wanner, R., Roth, H. M., Duhr, S., Baaske,
P., and Breitsprecher, D. (2014)
MicroScale Thermophoresis: Interaction
analysis and beyond. Journal of Molecular
Structure
Please note: This Application Note replaces NT-MO-025
because it contained erroneous information.
© 2015 NanoTemper Technologies GmbH
