All sedimentation experiments were collected on an Optima XL-A analytical ultracentrifuge using a An60Ti rotor (Beckman Coulter, Brea, CA). Sedimentation velocity experiments with 2 μM Cy3-labeled DNA were performed using Epon charcoal-filled double-sector centerpieces at 55,000 rpm with 0.03 cm spacing and recording scans every 8 min at 545 nm. Sedimentation equilibrium experiments were performed using Epon charcoal-filled six-sector centerpieces at the appropriate rpm with 0.001 cm spacing, scanned every 4 h, averaged from 10 replicates and recorded at 545 nm. Sedimentation velocity and equilibrium data were processed and analyzed with SedFit/SedPhat (Peter Schuck). The apparent molecular weights of the complexes were determined as described (25 ).
An60ti
Manufactured by Beckman Coulter
Sourced in United Kingdom, United States
The An60Ti is a fixed-angle rotor designed for high-speed centrifugation applications. It is compatible with Beckman Coulter centrifuges and can achieve a maximum speed of 60,000 rpm.
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Lab products found in correlation
Proteomelab xl 1, by Beckman Coulter (4 mentions)
Optima xl a, by Beckman Coulter (3 mentions)
Optima xl 1, by Beckman Coulter (3 mentions)
An50ti, by Beckman Coulter (1 mentions)
Sd75 16 600 column, by GE Healthcare (1 mentions)
Proteomelab xl 1 analytical ultracentrifuge, by Beckman Coulter (1 mentions)
Optima l 90k, by Beckman Coulter (1 mentions)
Flame s xr1 es spectrometer, by OceanOptics (1 mentions)
Flame s spectrometer, by OceanOptics (1 mentions)
Proteomelab xl a xl 1, by Beckman Coulter (1 mentions)
Proteomelab xl 1 protein characterization system, by Beckman Coulter (1 mentions)
Fit2d, by Wavemetrics (1 mentions)
Igor pro, by Wavemetrics (1 mentions)
22 protocols using an60ti
1
Analytical Ultracentrifugation Characterization
2
Sedimentation Velocity Analysis of Protein Complexes
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AUC sedimentation velocity experiments were performed at the Research Complex at Harwell (Harwell, UK) using a Beckman Coulter ProteomeLab XL-I ultracentrifuge and An-50Ti or An-60Ti rotors (Beckman Coulter) with sapphire window inserts. Experiments were conducted at 15 °C and 35,000 rpm with data recorded simultaneously at a 280 nm wavelength and via interference optics over 250 complete scans. Protein samples were at 50 mm concentration in 150 mm NaCl, 20 mm HEPES buffer, pH 7.5, with 2 mm Tris(2-carboxyethyl)phosphine and supplemented with 5% v/v DMSO or small molecule compounds at 2 mm final concentration as indicated. Data were analyzed using Sedfit (45 (link)).
3
Sedimentation Velocity Analysis of Purified Protein
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Sedimentation velocity experiments were used to determine the S value and molecular weight of the purified recombinant protein samples at 20°C on a Beckman XL-A analytical ultracentrifuge equipped with absorbance optics and an An60-Ti rotor. The samples were diluted to 1 OD at 280 nm. PBS was used as the reference buffer. The rotor speed was set at 35,000 rpm for these samples. The sedimentation coefficient was obtained using the c(s) method with the Sedfit software, which was kindly provided by Dr. P. Schuck at the National Institutes of Health (http://www.analyticalultracentrifugation.com ). And the molecular weight was calculated using the c(M) module of the Sedfit software.
4
Analytical Ultracentrifugation of Ptch1 Protein
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Analytical ultracentrifugation sedimentation velocity (AUC-SV) experiments were performed using a Beckman Coulter XL-I analytical ultracentrifuge equipped with a four-cell An-60 Ti analytical rotor. Four hundred μl protein sample from size-exclusion chromatography in the buffer containing 25 mM Tris pH 8.0, 150 mM NaCl, and 0.02% GDN and 400 μl buffer (25 mM Tris PH 8.0, 150 mM NaCl) were loaded into the sample sector and reference sector separately. The rotor with the cells was pre-scanned at a rotor speed of 70 × g for sample leakage. The run was started once the rotor temperature reached the set point at 20 °C. Sedimentation profiles were recorded by UV detector at 280 nm and Interference laser at 655 nm and scanned every 6 min.
Data were analyzed with GUSSI. The partial specific volume and dn/dc value for the protein are 0.74 cm3/g and 0.1896 cm3/g, respectively, according to the report. The extinction coefficient for Ptch1 is 1.27 L/(g cm), which was calculated with ProtParam on the ExPasy server. For GDN, the partial specific volume was measured by Density Meter as 0.80 cm3/g and dn/dc value was measured by multi-angle light scattering as 0.135 cm3/g.
Data were analyzed with GUSSI. The partial specific volume and dn/dc value for the protein are 0.74 cm3/g and 0.1896 cm3/g, respectively, according to the report. The extinction coefficient for Ptch1 is 1.27 L/(g cm), which was calculated with ProtParam on the ExPasy server. For GDN, the partial specific volume was measured by Density Meter as 0.80 cm3/g and dn/dc value was measured by multi-angle light scattering as 0.135 cm3/g.
5
Sedimentation Equilibrium Analysis of rGpA
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Sedimentation equilibrium experiments were carried out in a Beckman/Coulter XL-A analytical ultracentrifuge (Beckman/Coulter, Palo Alto, CA) using an An60Ti rotor at 10°C. Absorbance measurements at a wavelength of 266 nm for three independent replicates at three concentrations of 5 μΜ, 7 μM, and 8 µM were obtained at speeds of 6,000 rpm (6K), 7K, and 8K. A partial specific volume for fully glycosylated rGpA of 0.693633 was calculated by using the software program Sednterp (55 ), and a global fit analysis was performed in the program Ultrascan II, version 9.9 (56 (link)). The molecular mass reported is the mean ± standard deviation for the three independent replicates at all speeds analyzed.
6
Analytical Ultracentrifugation of Paratox
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Paratox was buffer exchanged into 20 mM Tris pH 7.5 100 mM NaCl 1 mM β-ME by gel-filtration using an SD75 16/600 column (GE Healthcare) then prepared as three samples at 219 μM, 329 μM, and 545 μM for velocity sedimentation experiments. The samples and buffer blanks were loaded into an AN-60Ti with double-channel centerpieces (Beckman Coulter). Data was collected using a ProteomeLab XL-1 (Beckman Coulter) at 50,000 rpm and 20 °C with 1 scan per minute at 280 nm in absorbance mode. A total of 370 scans were collected for each sample. Data was analyzed using SEDFIT and a continuous distribution model69 (link). For data fitting the buffer density and viscosity were calculated using SENTERP to be 1.00293 and 0.0169 poise respectively, and vbar for paratox to be 0.744 mL/g.
7
Sedimentation Velocity Analysis of gp32 and ssDNA
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Sedimentation velocity experiments were performed using the Beckman ProteomeLab XL-I Analytical Ultracentrifuge and sedimentation data were collected using the interference optics system. All protein and nucleic acid samples were dialyzed extensively against reference buffer, consisting of 10 mM HEPES (pH 7.5) and 30 mM KOAc, prior to centrifugation. The concentrations of both proteins and nucleic acids were re-measured after dialysis to ensure accurate final values. In each experiment 400 μl of sample and 400 μl of reference buffer were loaded, respectively, into the sample and reference sectors of a 1.2 cm double-sector Epon centerpiece and sedimented in a Beckman An60Ti ultracentrifuge rotor. Unless otherwise stated, experiments were performed at 20°C and at a rotor speed of 50 000 rpm. A partial specific volume ( ) of 0.732 ml/gm for gp32 protein was calculated from the amino acid composition of the protein (34 (link)). The free ssDNA constructs diffused too rapidly to form a discrete sedimentation boundary that could pull away from the meniscus and binding of the ssDNA constructs did not measurably alter the sedimentation coefficients of the gp32 components. All data analysis for the sedimentation velocity experiments was performed using the SedFit program (35 (link)–37 (link)).
8
Analytical Ultracentrifugation of PvS1 Proteins
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Prior to AUC analysis, protein samples were dialyzed against buffer pH 8 (50 mM Tris pH 8, 150 mM NaCl, and 10 mM CaCl2) or buffer pH 5 (50 mM NaCit pH 5, 150 mM NaCl, 10 mM CaCl2) at 4°C for 24 h. Protein samples were centrifuged in a Beckman Coulter XL-I analytical ultracentrifuge at 20°C in a four-hole rotor AN60-Ti equipped with 12 mm six sectors centerpieces. Detection of the protein concentration as a function of radial position and time was performed by optical density measurements at 280 nm. For sedimentation equilibrium experiments, 120 µL of PvS1FL or PvS1ΔBelt (2, 5, and 10 µM) in buffer pH 8 or buffer pH 5 were spun sequentially at rotor speeds of 9,000, 13,000, and 16,000 rpm. The data were acquired after reaching equilibrium for 1 h at each speed. The following parameters were calculated using Sednterp 1.09 and used for the analysis of the experiment: partial specific volume of 0.732 mL g−1, viscosity η = 1.068 cP, and density ρ = 1.0139 g mL−1.
Sedimentation equilibrium radial distributions were analyzed by global fitting using the single-species model with Ultrascan 9.5 software (http://www.ultrascan.uthscsa.edu ) (75 ). Monte Carlo analysis was performed on the fit in order to estimate measurement error.
Sedimentation equilibrium radial distributions were analyzed by global fitting using the single-species model with Ultrascan 9.5 software (
9
Sedimentation Velocity Analysis of RBPMS Variants
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An Optima XL-I (Beckman Coulter) centrifuge using an An60 Ti eight-hole rotor was used to measure sedimentation velocity (SV). Standard 12 mm double-sector Epon centerpieces with sapphire windows contained 400 μl of wild-type recombinant RBPMS-A or the mutant variants that were buffer exchanged with ultracentrifugation buffer (20 mM HEPES, 500 mM KCl, 1 mM TCEP, pH adjusted to 7.9) and adjusted to 0.5 mg/ml. Interference data were acquired at 40 000 rpm, 20°C, overnight. Ten scans were overlaid as a single point to reduce file size. The partial specific volume of the protein ( = 0.73 ml/g) and the viscosity and density of the buffer (η = 1.01036 × 10−2 mPa⋅s; ρ = 1.0232 g/ml) were calculated using SEDNTERP (Laue et al., 1992). One hundred scans taken over 10 h were used to determine the multi-component sedimentation coefficient c(s) distributions by direct boundary modeling of the Lamm equation using SEDFIT (v14.1) (17 (link)). The proportion of dimers to oligomers was determined by integration of the peaks, which were identified by their fitted mass.
10
Metal-Dependent Dimerization of rHC1 Protein
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The metal ion dependence of rHC1 dimerization was analyzed using both velocity and equilibrium AUC. All AUC experiments were conducted at 20 °C on a Beckman XL-A ultracentrifuge with an An60Ti rotor.
For velocity AUC, 18 μm WT rHC1 protein was prepared in HEPES-buffered saline, pH 7.5, in the presence of either 2.5 mm EDTA or 5 mm MgCl2. The samples were analyzed at 40,000 rpm for 5 h, with scans taken at 280 nm every 90 s. This experiment was conducted in triplicate with representative data shown in Fig. 3 A. Sedimentation coefficient distributions (c(s)) were calculated using SEDFIT (80 (link)).
For equilibrium AUC, measurements were made at three different concentrations of rHC1 (4, 11, and 22 μm ), and each of these were prepared with five different concentrations of MgCl2 (0 (2.5 mm EDTA), 0.1, 0.5, 1, and 5 mm ). Rotor speeds of 10,000, 15,000, and 20,000 rpm were used with scans at 280 nm (and 290 nm for the highest concentration) after equilibrium had been reached (18 h). The data (from triplicate experiments) were analyzed by global analysis with SEDFIT/SEDPHAT (81 (link)) and fitted to a monomer–dimer model.
For velocity AUC, 18 μ
For equilibrium AUC, measurements were made at three different concentrations of rHC1 (4, 11, and 22 μ
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