2.2 Proteins
1. SSB was purified to homogeneity as described before [6]. An
extinction coefficient of 113,000 M
À1 cm
À1 at 280 nm [24] is
used for calculation of molar concentrations, which are given as
tetramers.
2. The clamp loader used in this study (subunit composition
τ 3 δδ
0
χψ) contains an ψ-MBP variant carrying a SrtA cleavage
site (Fig. 3a) for site-specific labeling using a SrtA-mediated
reaction [17]. It was purified to homogeneity as described for
the τ 3 δδ
0
χψ clamp loader [25].
3. BSA was purchased in 98% purity, further purified by sizeexclusion chromatography, and used as a 100 μM stock solution. It serves as a carrier protein to avoid sticking of FITClabeled clamp loader (see Note 1).
4. StrA was purified as described elsewhere [26].
All proteins were flash-frozen in liquid N 2 in aliquots of at most
100 μL and stored at À80
C until use.
2.3 Oligonucleotides
DNA oligonucleotides (Table 1) used in this study were purchased
in HPLC quality. By annealing of the appropriate 30-mer A or B,
respectively, to a 55-mer or an 80-mer oligonucleotide, the template/primer constructs t/p 55/30 and t/p 80/30 were obtained
that consist of 30 nucleotides of double-stranded DNA with a
5
0 -overhang comprising 25 or 50 nucleotides, respectively
(Fig. 4a).
2.4 Peptide
FITC-peptide NH 2 -GGGK(FITC)AA-COOH was purchased and
carries a fluorescein isothiocyanate (FITC) probe at the lysine
residue. This peptide is used to introduce a fluorescein probe at
the C-terminus of the ψ subunit of the clamp loader by a SrtAmediated labeling reaction (Fig. 3a). An extinction coefficient of
70,000 M
À1 cm
À1 at 493 nm is used to determine the molar
concentration of the FITC-peptide [27].
2.5 Instrumentation
1. ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter, USA).
2. A fluorescence detection system with λ Ex ¼ 488 nm and
λ Em ¼ 505–565 nm (FDS, Aviv Biomedical, USA).
3. An FDS calibration cell required for radial and angular calibration filled with a solution of 100 nM fluorescein in 10 mM TrisHCl and 100 mM NaCl, pH 8.3 (see Note 2).
4. 8-hole An-50 Ti rotor.
5. 3 mm two-channel charcoal-filled Epon centerpieces (e.g.,
from Beckman Coulter, USA).
6. Standard AUC cell housings (e.g., from Beckman Coulter,
USA) or special fluorescence housings (e.g., from Nanolytics
Analysis of Protein-DNA Interactions by AUC
409
1. SSB was purified to homogeneity as described before [6]. An
extinction coefficient of 113,000 M
À1 cm
À1 at 280 nm [24] is
used for calculation of molar concentrations, which are given as
tetramers.
2. The clamp loader used in this study (subunit composition
τ 3 δδ
0
χψ) contains an ψ-MBP variant carrying a SrtA cleavage
site (Fig. 3a) for site-specific labeling using a SrtA-mediated
reaction [17]. It was purified to homogeneity as described for
the τ 3 δδ
0
χψ clamp loader [25].
3. BSA was purchased in 98% purity, further purified by sizeexclusion chromatography, and used as a 100 μM stock solution. It serves as a carrier protein to avoid sticking of FITClabeled clamp loader (see Note 1).
4. StrA was purified as described elsewhere [26].
All proteins were flash-frozen in liquid N 2 in aliquots of at most
100 μL and stored at À80
C until use.
2.3 Oligonucleotides
DNA oligonucleotides (Table 1) used in this study were purchased
in HPLC quality. By annealing of the appropriate 30-mer A or B,
respectively, to a 55-mer or an 80-mer oligonucleotide, the template/primer constructs t/p 55/30 and t/p 80/30 were obtained
that consist of 30 nucleotides of double-stranded DNA with a
5
0 -overhang comprising 25 or 50 nucleotides, respectively
(Fig. 4a).
2.4 Peptide
FITC-peptide NH 2 -GGGK(FITC)AA-COOH was purchased and
carries a fluorescein isothiocyanate (FITC) probe at the lysine
residue. This peptide is used to introduce a fluorescein probe at
the C-terminus of the ψ subunit of the clamp loader by a SrtAmediated labeling reaction (Fig. 3a). An extinction coefficient of
70,000 M
À1 cm
À1 at 493 nm is used to determine the molar
concentration of the FITC-peptide [27].
2.5 Instrumentation
1. ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter, USA).
2. A fluorescence detection system with λ Ex ¼ 488 nm and
λ Em ¼ 505–565 nm (FDS, Aviv Biomedical, USA).
3. An FDS calibration cell required for radial and angular calibration filled with a solution of 100 nM fluorescein in 10 mM TrisHCl and 100 mM NaCl, pH 8.3 (see Note 2).
4. 8-hole An-50 Ti rotor.
5. 3 mm two-channel charcoal-filled Epon centerpieces (e.g.,
from Beckman Coulter, USA).
6. Standard AUC cell housings (e.g., from Beckman Coulter,
USA) or special fluorescence housings (e.g., from Nanolytics
Analysis of Protein-DNA Interactions by AUC
409