method, the experimental sedimentation data are matched with the
best possible combination of sedimentation patterns from particles
of different sizes and in the resulting distribution of s-values the
contribution of the diffusional spread is eliminated [19]. This is
especially useful if both reaction partners contribute to the detected
signal since the diffusion-correction usually allows a separation
between the sedimentation coefficients of free A and the reaction
boundary. In cases like ours, where the faster sedimenting FITClabeled clamp loader is detected, but not the slower sedimenting
unlabeled SSB-saturated template/primer DNA, only the sedimentation of the reaction boundary can be observed (Fig. 5a). Even in
such cases, it is advantageous to use c(s) analysis since determination
of s rb by integration of the c(s) distribution in the range from s B to
s AB will omit the contribution of traces of free dye, aggregates, or
impurities to the obtained s-value.
In order to assess the affinity of the τ clamp loader to
SSB-saturated template/primer, we titrated a constant amount of
the faster sedimenting FITC-labeled clamp loader with increasing
excesses of unlabeled SSB-saturated template/primer. We used this
kind of titration since we wanted to impede the formation of higher
complexes, as one SSB tetramer can bind up to four χ subunits
[6, 7]. Otherwise, further clamp loader molecules could bind to the
DNA/SSB-complex via interactions solely with the SSB
C-terminus. Although this interaction is expected to be much
weaker, it could interfere with the analysis. Furthermore, we
could then investigate the effect of different unlabeled-primer/
template sequences on the affinity.
2 Materials
2.1 Buffers
and Solutions
All buffer solutions were prepared with chemicals of the highest
purity available in ultrapure H 2 O; pH was adjusted at room
temperature.
1. Buffer A: 20 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl 2 ,
1 mM TCEP, pH 7.5.
2. Buffer B: 20 mM Tris-HCl, 150 mM NaCl, 1 mM TCEP,
pH 7.5.
3. Buffer C: 20 mM Tris-HCl, 150 mM NaCl, pH 7.5.
4. ATP stock: 100 mM, pH 7.5.
5. TCEP stock: 0.5 M, pH 7.0.
6. MgCl 2 stock: 1 M.
7. CaCl 2 stock: 0.2 M.
8. Calibration solution: 100 nM fluorescein, 10 mM Tris-HCl,
100 mM NaCl, pH 8.3.
408
Andrea Bogutzki and Ute Curth
best possible combination of sedimentation patterns from particles
of different sizes and in the resulting distribution of s-values the
contribution of the diffusional spread is eliminated [19]. This is
especially useful if both reaction partners contribute to the detected
signal since the diffusion-correction usually allows a separation
between the sedimentation coefficients of free A and the reaction
boundary. In cases like ours, where the faster sedimenting FITClabeled clamp loader is detected, but not the slower sedimenting
unlabeled SSB-saturated template/primer DNA, only the sedimentation of the reaction boundary can be observed (Fig. 5a). Even in
such cases, it is advantageous to use c(s) analysis since determination
of s rb by integration of the c(s) distribution in the range from s B to
s AB will omit the contribution of traces of free dye, aggregates, or
impurities to the obtained s-value.
In order to assess the affinity of the τ clamp loader to
SSB-saturated template/primer, we titrated a constant amount of
the faster sedimenting FITC-labeled clamp loader with increasing
excesses of unlabeled SSB-saturated template/primer. We used this
kind of titration since we wanted to impede the formation of higher
complexes, as one SSB tetramer can bind up to four χ subunits
[6, 7]. Otherwise, further clamp loader molecules could bind to the
DNA/SSB-complex via interactions solely with the SSB
C-terminus. Although this interaction is expected to be much
weaker, it could interfere with the analysis. Furthermore, we
could then investigate the effect of different unlabeled-primer/
template sequences on the affinity.
2 Materials
2.1 Buffers
and Solutions
All buffer solutions were prepared with chemicals of the highest
purity available in ultrapure H 2 O; pH was adjusted at room
temperature.
1. Buffer A: 20 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl 2 ,
1 mM TCEP, pH 7.5.
2. Buffer B: 20 mM Tris-HCl, 150 mM NaCl, 1 mM TCEP,
pH 7.5.
3. Buffer C: 20 mM Tris-HCl, 150 mM NaCl, pH 7.5.
4. ATP stock: 100 mM, pH 7.5.
5. TCEP stock: 0.5 M, pH 7.0.
6. MgCl 2 stock: 1 M.
7. CaCl 2 stock: 0.2 M.
8. Calibration solution: 100 nM fluorescein, 10 mM Tris-HCl,
100 mM NaCl, pH 8.3.
408
Andrea Bogutzki and Ute Curth