and measurement, 0.1% (v/v) Tween 20 is added to the buffer
used for both serial dilution of NAG3 and dilution of HEWL
from the stock solution.
6. In case it proves important for reproducibility, it should be
noted that all of the data presented here were acquired using
reagents from Sigma Aldrich (HEWL: L4919, NAG3: T2144).
The IUPAC name for NAG3 is N
0 N
00 N
000 -triacetyl chitotriose,
but it is also known by several other names listed at Pubchem
under the compound ID 123774.
7. When ordering multiple vials of SYPRO Orange, consistency of
fluorescence intensity over time can be improved by pooling
and mixing the aliquots and storing the result in a black Eppendorf tube at À20
C.
8. We use a single-beam instrument and a 1-cm pathlength
cuvette with a fill volume of 200 μL, which gives acceptable
reproducibility with relatively little sample consumption.
9. This simple and readily employed correction [25] is a linearization of the inverse fourth power dependence of scattering upon
wavelength, appropriate for any protein without cofactor
absorbance at 333 nm. If the solutions are prepared correctly,
the scattering correction should be very small (<5% of the total
absorbance at 280 nm).
10. This is essentially identical to the experimentally determined
value (37,789 M
À1 cm
À1 ) [25].
11. If weighing proves unreliable for the determination of the
NAG3 concentration, another (albeit less satisfactory
approach) would be to use several replicate ITC experiments
on a NAG3 stock to benchmark the concentration on the
assumption that the lysozyme concentration can be accurately
and reproducibly determined by spectrophotometry. It would
also be possible to determine the concentration from peak
integrals in a 1D NMR experiment, though we have not
attempted to do so.
12. This experiment is designed to show how the thermal shift
assay can be used as an initial high-throughput screen for
binding. In those circumstances, it is usual to use ligand concentrations that are sufficiently high to ensure saturation of
binding for a wide range of dissociation constants, but not so
high as to cause shifts in pH of the experimental buffer.
13. Since the fluorescence intensity from SYPRO Orange usually
increases in the presence of unfolded protein, it is not possible
to optimize the detector gain setting from the initial fluorescence. Instead, an appropriate value must be determined
empirically for each instrument (and sometimes for a new
batch of dye) in a test experiment.
72
Xiaochun Li-Blatter et al.
used for both serial dilution of NAG3 and dilution of HEWL
from the stock solution.
6. In case it proves important for reproducibility, it should be
noted that all of the data presented here were acquired using
reagents from Sigma Aldrich (HEWL: L4919, NAG3: T2144).
The IUPAC name for NAG3 is N
0 N
00 N
000 -triacetyl chitotriose,
but it is also known by several other names listed at Pubchem
under the compound ID 123774.
7. When ordering multiple vials of SYPRO Orange, consistency of
fluorescence intensity over time can be improved by pooling
and mixing the aliquots and storing the result in a black Eppendorf tube at À20
C.
8. We use a single-beam instrument and a 1-cm pathlength
cuvette with a fill volume of 200 μL, which gives acceptable
reproducibility with relatively little sample consumption.
9. This simple and readily employed correction [25] is a linearization of the inverse fourth power dependence of scattering upon
wavelength, appropriate for any protein without cofactor
absorbance at 333 nm. If the solutions are prepared correctly,
the scattering correction should be very small (<5% of the total
absorbance at 280 nm).
10. This is essentially identical to the experimentally determined
value (37,789 M
À1 cm
À1 ) [25].
11. If weighing proves unreliable for the determination of the
NAG3 concentration, another (albeit less satisfactory
approach) would be to use several replicate ITC experiments
on a NAG3 stock to benchmark the concentration on the
assumption that the lysozyme concentration can be accurately
and reproducibly determined by spectrophotometry. It would
also be possible to determine the concentration from peak
integrals in a 1D NMR experiment, though we have not
attempted to do so.
12. This experiment is designed to show how the thermal shift
assay can be used as an initial high-throughput screen for
binding. In those circumstances, it is usual to use ligand concentrations that are sufficiently high to ensure saturation of
binding for a wide range of dissociation constants, but not so
high as to cause shifts in pH of the experimental buffer.
13. Since the fluorescence intensity from SYPRO Orange usually
increases in the presence of unfolded protein, it is not possible
to optimize the detector gain setting from the initial fluorescence. Instead, an appropriate value must be determined
empirically for each instrument (and sometimes for a new
batch of dye) in a test experiment.
72
Xiaochun Li-Blatter et al.