3.3 Fluorescence
Intensity
3.3.1 Preparation
of Titration
for Fluorescence Intensity
The aim is to make a 24-point titration of NAG3 against a constant
concentration of HWEL, with an appropriate range and pointdensity for accurate fitting of the dissociation constant (K d ).
1. Make a 4-μM solution of HEWL and a 450-μM solution of
NAG3, both in the standard assay buffer, according to the
method outlined in Subheading 3.1.
2. Choose one row of a 384-well microplate and dispense 80 μL
of the 450 μM NAG3 solution into the first well of that row.
3. Dispense 20 μL of the standard assay buffer into the remaining
23 wells of that row.
4. Take 60 μL of solution from the first well in the row (containing the NAG3 solution), touching the tip on the inside of the
well as you remove it, to remove residual solution on the
outside of the tip.
5. Pipette the solution into the second well in the row (which
should already contain 20 μL of buffer). Mix at least 4 times,
aspirating at least two-thirds of the total solution in the well.
Keep the pipette tip under the meniscus at all times to prevent
air bubbles. Touch the tip on the inside of the well as you
withdraw it.
6. Change the pipette tip and transfer 60 μL of the mixed solution
in the second well into the third well. Mix, then repeat this
process for the remaining wells.
7. Remove 60 μL of solution from the final well in the row so that
all wells contain 20 μL of solution.
8. This will produce a 24-point serial dilution of NAG3 with the
dilution ratio of 3:1 (a dilution factor at each step of 1.33).
9. Now add 50 μL of the 4 μM HEWL solution to each well of the
row containing the NAG3 solutions. Mix the solution well (see
step 5 above).
10. This will give a 24-point titration with a varying concentration
of NAG3 and a constant concentration of 2.85 μM HEWL in a
working volume of 70 μL (see Note 17).
To obtain a more reliable estimate of the K d , with more realistic
errors, you could include one or more replicate titrations in other
rows of the same plate. It is important to repeat the serial dilution
for the replicate titration so that the serial dilution errors are
accounted for in the error estimates for the fitted parameters.
3.3.2 Fluorescence
Intensity Measurement
1. Incubate the titration at the measurement temperature for
10 min prior to measurement. Place a lid on the microplate
to avoid sample evaporation.
Interactions by Multiple Methods
57
Intensity
3.3.1 Preparation
of Titration
for Fluorescence Intensity
The aim is to make a 24-point titration of NAG3 against a constant
concentration of HWEL, with an appropriate range and pointdensity for accurate fitting of the dissociation constant (K d ).
1. Make a 4-μM solution of HEWL and a 450-μM solution of
NAG3, both in the standard assay buffer, according to the
method outlined in Subheading 3.1.
2. Choose one row of a 384-well microplate and dispense 80 μL
of the 450 μM NAG3 solution into the first well of that row.
3. Dispense 20 μL of the standard assay buffer into the remaining
23 wells of that row.
4. Take 60 μL of solution from the first well in the row (containing the NAG3 solution), touching the tip on the inside of the
well as you remove it, to remove residual solution on the
outside of the tip.
5. Pipette the solution into the second well in the row (which
should already contain 20 μL of buffer). Mix at least 4 times,
aspirating at least two-thirds of the total solution in the well.
Keep the pipette tip under the meniscus at all times to prevent
air bubbles. Touch the tip on the inside of the well as you
withdraw it.
6. Change the pipette tip and transfer 60 μL of the mixed solution
in the second well into the third well. Mix, then repeat this
process for the remaining wells.
7. Remove 60 μL of solution from the final well in the row so that
all wells contain 20 μL of solution.
8. This will produce a 24-point serial dilution of NAG3 with the
dilution ratio of 3:1 (a dilution factor at each step of 1.33).
9. Now add 50 μL of the 4 μM HEWL solution to each well of the
row containing the NAG3 solutions. Mix the solution well (see
step 5 above).
10. This will give a 24-point titration with a varying concentration
of NAG3 and a constant concentration of 2.85 μM HEWL in a
working volume of 70 μL (see Note 17).
To obtain a more reliable estimate of the K d , with more realistic
errors, you could include one or more replicate titrations in other
rows of the same plate. It is important to repeat the serial dilution
for the replicate titration so that the serial dilution errors are
accounted for in the error estimates for the fitted parameters.
3.3.2 Fluorescence
Intensity Measurement
1. Incubate the titration at the measurement temperature for
10 min prior to measurement. Place a lid on the microplate
to avoid sample evaporation.
Interactions by Multiple Methods
57