absorbance must be corrected for contributions of the FITC
dye after measuring the absorbance of the labeled protein at
493 nm:
A
protein
280
¼ A 280 À 0:3 Á A 493
ð5Þ
7. Calculate the degree of labeling for the FITC-labeled clamp
loader from the concentration of the FITC dye divided by the
concentration of the protein. The concentration of the
attached FITC dye can be calculated from the absorbance at
493 nm using an extinction coefficient of 70,000 M
À1 cm
À1 .
Using this labeling protocol, the degree of labeling is typically
0.85–0.9.
8. Pool fractions containing high amounts of FITC-labeled clamp
loader with a degree of labeling of at least 0.85, flash-freeze
aliquots of 10–20 μL in liquid N 2 , and store at À80
C until
use. The labeled clamp loader typically has a concentration of
1.5–2 μM.
9. Verify the homogeneity of the FITC-clamp loader preparation
by AUC sedimentation velocity experiments in buffer A at
40,000 rpm using the fluorescence detection system (as shown
in Fig. 2).
3.2 Template/Primer
Preparation
1. Determine the concentrations of oligonucleotides by UV spectroscopy at 260 nm using extinction coefficients calculated
from nucleotide composition using IDT SciTools [31]:
290,800 M
À1 cm
À1 for 30-mer A, 510,500 M
À1 cm
À1 for
55-mer, 281,600 M
À1
cm
À1
for 30-mer B, and
765,100 M
À1 cm
À1 for 80-mer.
2. Mix 15 μM of template (55-mer or 80-mer) with 25 μM of the
appropriate primer (30-mer A or B, respectively) in a total
volume of 550 μL of buffer C.
3. Heat the mixture to 90
C for 10 min and let cool down at
room temperature.
4. To separate hybridized template/primer from excess primer,
apply the DNA solution to a Superdex 200 Increase 10/300
GL column (GE Healthcare, Germany) equilibrated with
buffer C at a flow rate of 0.5 mL/min (as shown in Fig. 4b).
5. Determine the concentrations of template/primer solutions by
UV spectroscopy using extinction coefficients at 260 nm calculated from nucleotide composition using IDT SciTools [31]:
703,707 M
À1 cm
À1 for t/p 55/30 and 954,221 M
À1 cm
À1 for
t/p 80/30.
Analysis of Protein-DNA Interactions by AUC
411
dye after measuring the absorbance of the labeled protein at
493 nm:
A
protein
280
¼ A 280 À 0:3 Á A 493
ð5Þ
7. Calculate the degree of labeling for the FITC-labeled clamp
loader from the concentration of the FITC dye divided by the
concentration of the protein. The concentration of the
attached FITC dye can be calculated from the absorbance at
493 nm using an extinction coefficient of 70,000 M
À1 cm
À1 .
Using this labeling protocol, the degree of labeling is typically
0.85–0.9.
8. Pool fractions containing high amounts of FITC-labeled clamp
loader with a degree of labeling of at least 0.85, flash-freeze
aliquots of 10–20 μL in liquid N 2 , and store at À80
C until
use. The labeled clamp loader typically has a concentration of
1.5–2 μM.
9. Verify the homogeneity of the FITC-clamp loader preparation
by AUC sedimentation velocity experiments in buffer A at
40,000 rpm using the fluorescence detection system (as shown
in Fig. 2).
3.2 Template/Primer
Preparation
1. Determine the concentrations of oligonucleotides by UV spectroscopy at 260 nm using extinction coefficients calculated
from nucleotide composition using IDT SciTools [31]:
290,800 M
À1 cm
À1 for 30-mer A, 510,500 M
À1 cm
À1 for
55-mer, 281,600 M
À1
cm
À1
for 30-mer B, and
765,100 M
À1 cm
À1 for 80-mer.
2. Mix 15 μM of template (55-mer or 80-mer) with 25 μM of the
appropriate primer (30-mer A or B, respectively) in a total
volume of 550 μL of buffer C.
3. Heat the mixture to 90
C for 10 min and let cool down at
room temperature.
4. To separate hybridized template/primer from excess primer,
apply the DNA solution to a Superdex 200 Increase 10/300
GL column (GE Healthcare, Germany) equilibrated with
buffer C at a flow rate of 0.5 mL/min (as shown in Fig. 4b).
5. Determine the concentrations of template/primer solutions by
UV spectroscopy using extinction coefficients at 260 nm calculated from nucleotide composition using IDT SciTools [31]:
703,707 M
À1 cm
À1 for t/p 55/30 and 954,221 M
À1 cm
À1 for
t/p 80/30.
Analysis of Protein-DNA Interactions by AUC
411