2.3 Other Reagents
l
DyLight488 NHS-ester dye: 1 Â 50 μg aliquot.
l
SYPRO Orange: 1 vial of 5000Â stock solution (see Note 7).
l
Biacore Amine Coupling Kit.
2.4 Instruments
l
Thermal shift assay: Differential scanning fluorimetry (DSF)
measurements on HEWL in the presence and absence of NAG3
should be performed using a thermocycler designed for quantitative polymerase chain reactions (qPCR). The instrument
should be capable of measuring fluorescence emission for the
dye SYPRO Orange (excitation maximum 492 nm, emission
maximum 610 nm) during a continuous thermal ramp. The
data presented here were measured using a Qiagen RotorGene Q, employing a combination of the green excitation and
orange emission channels for fluorescence detection. Examples
of other suitable instruments are given in Ref. 10. Samples
should be prepared and measured in PCR tubes or plates suited
to the instrument used.
l
Fluorescence intensity titration: The intensity of intrinsic fluorescence for titration of NAG3 vs. HEWL should be measured
with a plate reader with monochromators or filters allowing
measurement in the UV. The data presented here were measured
using a BioTek Synergy H1 plate reader, with an excitation
wavelength of 290 nm, an emission wavelength of 350 nm,
and a fixed bandwidth of 18 nm in excitation and emission
(manufacturer’s specification). Essentially identical results were
achieved with other plate readers (Tecan, Molecular Devices)
with similar settings. The samples for fluorescence intensity
measurements presented here were prepared and measured in
Corning nonbinding surface flat-bottom 384-well black microplates (e.g., Corning 3575). It is anticipated that other brands of
microplates with nonbinding surfaces ought to give equivalent
results.
l
MST: Measurements for titration of NAG3 vs. HEWL should
be performed using an instrument with fluorescence excitation
and emission optics that are suitable for the DyLight488 dye
(excitation maximum 493 nm, emission maximum 518 nm).
The data presented here were measured with a Nanotemper
Monolith NT.115 instrument using the blue channel excitation
and emission optics and standard capillaries. The samples for
MST measurements presented here were prepared in 200-μL
microtubes provided by Nanotemper, though it is anticipated
that other microtubes or microplates with a nonbinding surface
could be used.
l
ITC: Measurements should be made using a titrating power
compensation calorimeter with a suitable sensitivity and working
volume. The data presented here were measured using two
Interactions by Multiple Methods
53
l
DyLight488 NHS-ester dye: 1 Â 50 μg aliquot.
l
SYPRO Orange: 1 vial of 5000Â stock solution (see Note 7).
l
Biacore Amine Coupling Kit.
2.4 Instruments
l
Thermal shift assay: Differential scanning fluorimetry (DSF)
measurements on HEWL in the presence and absence of NAG3
should be performed using a thermocycler designed for quantitative polymerase chain reactions (qPCR). The instrument
should be capable of measuring fluorescence emission for the
dye SYPRO Orange (excitation maximum 492 nm, emission
maximum 610 nm) during a continuous thermal ramp. The
data presented here were measured using a Qiagen RotorGene Q, employing a combination of the green excitation and
orange emission channels for fluorescence detection. Examples
of other suitable instruments are given in Ref. 10. Samples
should be prepared and measured in PCR tubes or plates suited
to the instrument used.
l
Fluorescence intensity titration: The intensity of intrinsic fluorescence for titration of NAG3 vs. HEWL should be measured
with a plate reader with monochromators or filters allowing
measurement in the UV. The data presented here were measured
using a BioTek Synergy H1 plate reader, with an excitation
wavelength of 290 nm, an emission wavelength of 350 nm,
and a fixed bandwidth of 18 nm in excitation and emission
(manufacturer’s specification). Essentially identical results were
achieved with other plate readers (Tecan, Molecular Devices)
with similar settings. The samples for fluorescence intensity
measurements presented here were prepared and measured in
Corning nonbinding surface flat-bottom 384-well black microplates (e.g., Corning 3575). It is anticipated that other brands of
microplates with nonbinding surfaces ought to give equivalent
results.
l
MST: Measurements for titration of NAG3 vs. HEWL should
be performed using an instrument with fluorescence excitation
and emission optics that are suitable for the DyLight488 dye
(excitation maximum 493 nm, emission maximum 518 nm).
The data presented here were measured with a Nanotemper
Monolith NT.115 instrument using the blue channel excitation
and emission optics and standard capillaries. The samples for
MST measurements presented here were prepared in 200-μL
microtubes provided by Nanotemper, though it is anticipated
that other microtubes or microplates with a nonbinding surface
could be used.
l
ITC: Measurements should be made using a titrating power
compensation calorimeter with a suitable sensitivity and working
volume. The data presented here were measured using two
Interactions by Multiple Methods
53