3. Incubate the labeling reaction for 1 h at room temperature in a
foil-covered tube.
4. After incubation, dilute the labeling reaction solution to a total
of 2 mL volume using the labeling buffer.
5. Dialyze the labeling reaction solution against three changes of
300 mL of standard assay buffer using a dialysis cassette or
membrane with a nominal molecular weight cutoff of 10 kDa.
6. Measure the absorbance of the dialyzed labeling solution at
280 and 493 nm using fresh standard assay buffer as a reference. It should be possible to measure the absorbance directly
using a small-volume 1-cm pathlength cuvette.
7. Calculate the concentration of DyLight488 from the absorbance at 493 nm using the Beer–Lambert law and a molar
extinction coefficient of 70,000 M
À1 cm
À1 .
8. Calculate the contribution of DyLight488 to the measured
absorbance at 280 nm by multiplying the measured absorbance
at 493 nm by 0.147 (this factor is specific to the dye and
represents its relative absorbance at 280 nm compared to
493 nm).
9. Subtract this contribution from DyLight488 from the total
measured absorbance at 280 nm.
10. Use the remainder, corresponding to the absorbance from
HEWL at 280 nm, to calculate the HEWL concentration
using the Beer–Lambert law and a molar extinction coefficient
of 37,970 M
À1 cm
À1 .
11. Calculate the ratio of DyLight488:HEWL. This should be
approximately 1:1 (see Note 22).
12. The Dylight488 labeled HEWL (HEWL-D488) should be
dispensed into PCR tubes in 20-μL aliquots, cryo-cooled
using liquid nitrogen, and stored at À80
C. It has proved
stable under these conditions for more than 5 years.
3.4.2 Preparation
of Titration for MST
The aim is to make a 16-point titration of NAG against a constant
concentration of HEWL-D488, with an appropriate range and
point-density for accurate fitting of the dissociation constant (K d ).
1. Make 90 μL of 300 μM NAG3 in MST buffer in a PCR tube.
2. Using that 90 μL as the first point, make a 16-point, 2:1 serial
dilution from that stock. Follow a similar procedure to that
outlined in detail in Subheading 3.3.1, but in this case, mix
60 μL of the preceding higher concentration in the serial
dilution with 30 μL of buffer at each step. Remember to
remove 60 μL of solution from the final tube in the titration
series so that all tubes contain 30 μL of solution.
60
Xiaochun Li-Blatter et al.
foil-covered tube.
4. After incubation, dilute the labeling reaction solution to a total
of 2 mL volume using the labeling buffer.
5. Dialyze the labeling reaction solution against three changes of
300 mL of standard assay buffer using a dialysis cassette or
membrane with a nominal molecular weight cutoff of 10 kDa.
6. Measure the absorbance of the dialyzed labeling solution at
280 and 493 nm using fresh standard assay buffer as a reference. It should be possible to measure the absorbance directly
using a small-volume 1-cm pathlength cuvette.
7. Calculate the concentration of DyLight488 from the absorbance at 493 nm using the Beer–Lambert law and a molar
extinction coefficient of 70,000 M
À1 cm
À1 .
8. Calculate the contribution of DyLight488 to the measured
absorbance at 280 nm by multiplying the measured absorbance
at 493 nm by 0.147 (this factor is specific to the dye and
represents its relative absorbance at 280 nm compared to
493 nm).
9. Subtract this contribution from DyLight488 from the total
measured absorbance at 280 nm.
10. Use the remainder, corresponding to the absorbance from
HEWL at 280 nm, to calculate the HEWL concentration
using the Beer–Lambert law and a molar extinction coefficient
of 37,970 M
À1 cm
À1 .
11. Calculate the ratio of DyLight488:HEWL. This should be
approximately 1:1 (see Note 22).
12. The Dylight488 labeled HEWL (HEWL-D488) should be
dispensed into PCR tubes in 20-μL aliquots, cryo-cooled
using liquid nitrogen, and stored at À80
C. It has proved
stable under these conditions for more than 5 years.
3.4.2 Preparation
of Titration for MST
The aim is to make a 16-point titration of NAG against a constant
concentration of HEWL-D488, with an appropriate range and
point-density for accurate fitting of the dissociation constant (K d ).
1. Make 90 μL of 300 μM NAG3 in MST buffer in a PCR tube.
2. Using that 90 μL as the first point, make a 16-point, 2:1 serial
dilution from that stock. Follow a similar procedure to that
outlined in detail in Subheading 3.3.1, but in this case, mix
60 μL of the preceding higher concentration in the serial
dilution with 30 μL of buffer at each step. Remember to
remove 60 μL of solution from the final tube in the titration
series so that all tubes contain 30 μL of solution.
60
Xiaochun Li-Blatter et al.