Instruments, Germany) for AUC experiments using absorbance detection or fluorescence detection, respectively.
7. UV/VIS spectrometer such as the Jasco V-560.
8. Protein purification system such as the A ¨ KTApurifier
(GE Healthcare, Germany) equipped with an appropriate
size-exclusion chromatography column, e.g., Superdex
200 Increase 10/300 GL column (GE Healthcare, Germany).
2.6 Software
1. SEDFIT [23] is a program package for the analysis of AUC
data, was used to generate c(s) distributions from the raw data
of the AUC experiments, and is available for free at http://
analyticalultracentrifugation.com/download.htm.
2. GUSSI [28] is used for the generation of binding isotherms
from c(s) distributions and graph plotting. It is available as a
zipped archive for free at https://www.utsouthwestern.edu/
labs/mbr/software/.
3. SEDPHAT [29] used to analyze binding isotherms is available
for free at http://analyticalultracentrifugation.com/sedphat/
download.htm or at https://sedfitsedphat.nibib.nih.gov/soft
ware/default.aspx.
All programs should be extracted into the same folder, e.g., C:
\sedfit.
3 Methods
3.1 Labeling
of the Clamp Loader
1. Thaw the clamp loader-ψMBP on ice.
2. Dissolve an appropriate amount of FITC-peptide in buffer B.
3. Prepare the labeling reaction mixture in a total volume of
500 μL by mixing the components in the following order:
Buffer B, 0.5 mM FITC-peptide, 6 mM CaCl 2 , 10 μM clamp
loader-ψMBP, and 75 μM SrtA.
4. Following incubation at 25
C overnight in the dark, apply the
labeling reaction mixture to a Superdex 200 Increase 10/300
GL column (GE Healthcare, Germany), equilibrated with
buffer B supplemented with 1 μM BSA (see Note 3), at a flow
rate of 0.5 mL/min to separate FITC-labeled clamp loader
from MBP, SrtA, and nonreacted peptide (as shown in Fig. 3b).
5. To determine which fractions to pool, analyze the samples by
SDS-PAGE and visualize the proteins by Coomassie staining
and fluorescence imaging with excitation at 312 nm (as shown
in Fig. 3c).
6. Determine the concentration of purified complex by UV spectroscopy using an extinction coefficient of 298,510 M
À1 cm
À1
for the clamp loader at 280 nm [30]. Beforehand, the
410
Andrea Bogutzki and Ute Curth
7. UV/VIS spectrometer such as the Jasco V-560.
8. Protein purification system such as the A ¨ KTApurifier
(GE Healthcare, Germany) equipped with an appropriate
size-exclusion chromatography column, e.g., Superdex
200 Increase 10/300 GL column (GE Healthcare, Germany).
2.6 Software
1. SEDFIT [23] is a program package for the analysis of AUC
data, was used to generate c(s) distributions from the raw data
of the AUC experiments, and is available for free at http://
analyticalultracentrifugation.com/download.htm.
2. GUSSI [28] is used for the generation of binding isotherms
from c(s) distributions and graph plotting. It is available as a
zipped archive for free at https://www.utsouthwestern.edu/
labs/mbr/software/.
3. SEDPHAT [29] used to analyze binding isotherms is available
for free at http://analyticalultracentrifugation.com/sedphat/
download.htm or at https://sedfitsedphat.nibib.nih.gov/soft
ware/default.aspx.
All programs should be extracted into the same folder, e.g., C:
\sedfit.
3 Methods
3.1 Labeling
of the Clamp Loader
1. Thaw the clamp loader-ψMBP on ice.
2. Dissolve an appropriate amount of FITC-peptide in buffer B.
3. Prepare the labeling reaction mixture in a total volume of
500 μL by mixing the components in the following order:
Buffer B, 0.5 mM FITC-peptide, 6 mM CaCl 2 , 10 μM clamp
loader-ψMBP, and 75 μM SrtA.
4. Following incubation at 25
C overnight in the dark, apply the
labeling reaction mixture to a Superdex 200 Increase 10/300
GL column (GE Healthcare, Germany), equilibrated with
buffer B supplemented with 1 μM BSA (see Note 3), at a flow
rate of 0.5 mL/min to separate FITC-labeled clamp loader
from MBP, SrtA, and nonreacted peptide (as shown in Fig. 3b).
5. To determine which fractions to pool, analyze the samples by
SDS-PAGE and visualize the proteins by Coomassie staining
and fluorescence imaging with excitation at 312 nm (as shown
in Fig. 3c).
6. Determine the concentration of purified complex by UV spectroscopy using an extinction coefficient of 298,510 M
À1 cm
À1
for the clamp loader at 280 nm [30]. Beforehand, the
410
Andrea Bogutzki and Ute Curth