Beckman optima xl 1

Manufactured by Beckman Coulter

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The Beckman Optima XL-I is an analytical ultracentrifuge designed for the characterization of macromolecules and particles in solution. It utilizes sedimentation velocity and sedimentation equilibrium techniques to provide information about the size, shape, and interactions of various biomolecules.

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Beckman auc sample cells, by Beckman Coulter (3 mentions) 60 ti rotor, by Beckman Coulter (2 mentions) 4800 maldi tof tof instrument, by Thermo Fisher Scientific (1 mentions) Optima xl 1, by Beckman Coulter (1 mentions) Chirascan plus cd spectrometer, by Applied Photophysics (1 mentions) Anti60 rotor, by Beckman Coulter (1 mentions)

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Optima XL-I (230 citations) Beckman XL-I (16 citations)

22 protocols using beckman optima xl 1

Analytical Ultracentrifugation of Peptide Samples

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Sedimentation-velocity
(SV) experiments were conducted at 20 °C in a Beckman-Optima
XL-I analytical ultracentrifuge using an An-60 Ti rotor. Samples were
prepared in PBS at 140 μM total peptide concentration. Samples
were centrifuged at a speed of 50,000 or 60,000 rpm. The partial specific
volume (v̅) for each peptide was calculated
and data sets were fitted to a single, ideal species model using SEDFIT.
Sedimentation-equilibrium (SE) experiments were conducted at 20 °C
in a Beckman-Optima XL-I analytical ultracentrifuge using an An-50
Ti rotor. Samples were prepared in PBS at 70 μM total peptide
concentration. Samples were centrifuged at a range of speeds from
18,000–39,000 rpm with defined intervals, and 8 h between subsequent
scans. Data sets were analyzed using SEDPHAT.

Thompson H.F., Beesley J.L., Langlands H.D., Edgell C.L., Savery N.J, & Woolfson D.N. (2023). Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies. ACS Synthetic Biology, 12(4), 1308-1319.

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Analytical Ultracentrifugation of NEO1-NET1-RGMB Complexes

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Sedimentation velocity (SV) experiments were performed at 20 °C using a Beckman Optima XL-1 analytical ultracentrifuge (Beckman Instruments), utilising a scanning absorbance of 280 nm and interference optics. Samples were contained within 12 mm Epon sector-shaped two-channel centerpieces and spun at 400,000 rpm (An60Ti rotor, Beckman Coulter Inc., CA), with 100 sample distribution scans taken in 6 minute intervals, alongside interference optics. Absorbance data for scans were analyzed using the program SedFit (Brown and Schuck, 2006 (link)) for size-and-shape distributions [c(s,fr), where fr is the frictional ratio and for a sphere fr = 1 and for other species fr > 1](Brown and Schuck, 2006 (link)). This enables the plotting of contour plots of c(s,M), where M is the weight of the protein. In all cases, a partial specific volume value of 0.73 ml g^-1 was used.
For SV-AUC experiments, binary NEO1_FN456-NET1_ΔNTR and ternary NEO1_FN456-NET1_ΔNTR-RGMB_ECD complexes were assembled via mixing of components in equimolar ratios, using wild type NET1_ΔNTR or NET1_FL, and ‘Interface-1’ and ‘Interface-2’ mutants. Complexes were then dialyzed for 16 hours against a buffer containing 10 mM HEPES pH 7.5, 150 mM NaCl, 2 mM CaCl₂, and the concentration was subsequently adjusted to 3 mg/ml via dilution with dialysis buffer.

Robinson R.A., Griffiths S.C., van de Haar L.L., Malinauskas T., van Battum E.Y., Zelina P., Schwab R.A., Karia D., Malinauskaite L., Brignani S., van den Munkhof M.H., Düdükcü Ö., De Ruiter A.A., Van den Heuvel D.M., Bishop B., Elegheert J., Aricescu A.R., Pasterkamp R.J, & Siebold C. (2021). Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling. Cell, 184(8), 2103-2120.e31.

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Analytical Ultracentrifugation of LmTK Variants

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C-terminal histagged LmTK stabilized with dTTP was dialyzed against SEC buffer to remove dTTP and subsequently diluted with dialysis buffer to 0.15 mg·mL^-1 and 0.45 mg·mL^-1. C-terminal histagged LmTK stabilized with dThd and AppNHp was dialyzed against SEC buffer containing 1 mM dThd and 1 mM AppNHp and diluted in dialysis buffer to 0.15 mg·mL^-1 and 1.0 mg·mL^-1. Analytical ultracentrifugation on all samples was carried out with a Beckman Optima XL/1 (Beckman Coulter) using an AN50Ti rotor at 42 000 rpm and 20°C. Sedimentation was recorded using the refractive index with the respective dialysis buffers as reference.

Timm J., Bosch-Navarrete C., Recio E., Nettleship J.E., Rada H., González-Pacanowska D, & Wilson K.S. (2015). Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major. PLoS Neglected Tropical Diseases, 9(5), e0003781.

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Sedimentation-velocity of Astrin Complex

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Sedimentation-velocity experiments for Astrin 465–693 complex constructs were conducted using purified protein at ~2 µM (see Figure 1—figure supplement 1E) in modified Column Buffer (DTT replaced with 0.5 mM TCEP), and using a Beckman Optima XL-I analytical ultracentrifuge (Beckman Coulter, Indianapolis, IN) in absorbance mode (MIT Biophysical Instrumentation Facility, Cambridge, MA). Data were collected at 20°C at 30,000 rpm. The data were fit using SEDFIT with a continuous sedimentation coefficient distribution model, assuming a single frictional coefficient. Molecular weights were estimated using the best-fit frictional coefficients. We note that the Stokes radius predicted by this fit was inconsistent with that determined by gel filtration (slightly larger than the 8.5 nm standard). When using the AUC S-value and the approximate gel filtration Stokes radius, the calculated fit is closer to a complex with 4 SKAP subunits, but 2 each of the remaining subunits. We chose the 2:2:2:2 complex fit as gel filtration migration of elongated proteins is subject to some amount of error versus globular size standards. However, we cannot exclude the possibility that there are more than 2 SKAP subunits per complex.

Kern D.M., Monda J.K., Su K.C., Wilson-Kubalek E.M, & Cheeseman I.M. (2017). Astrin-SKAP complex reconstitution reveals its kinetochore interaction with microtubule-bound Ndc80. eLife, 6, e26866.

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Hydrodynamic Characterization of Proteins

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Hydrodynamic characterization of the proteins, dialysed against vesicles buffer plus 1% glycerol, was achieved by sedimentation velocity at 48,000 rpm with a protein concentration of 0.5 mg·mL⁻¹. Experiments were conducted in a Beckman Optima XL-I analytical ultracentrifuge (Beckman Coulter) and sedimentation coefficient distributions were calculated with SEDFIT71 (link).

Fernández C., Núñez-Ramírez R., Jiménez M., Rivas G, & Giraldo R. (2016). RepA-WH1, the agent of an amyloid proteinopathy in bacteria, builds oligomeric pores through lipid vesicles. Scientific Reports, 6, 23144.

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Sedimentation Velocity Analysis of bpAFP

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A Beckman Optima XL-I Analytical ultracentrifuge (Beckman Coulter) was used for sedimentation velocity measurements at 40,000 rpm at 4 °C with 2.5 mg of bpAFP in 0.5 mL AFP of 10 mM sodium phosphate (pH 7.9). Double sector charcoal-Epon cells equipped with quartz windows were used and concentration distributions were determined by absorbance optics. Sedimentation coefficient distributions were determined using standard methodology

Mahatabuddin S., Hanada Y., Nishimiya Y., Miura A., Kondo H., Davies P.L, & Tsuda S. (2017). Concentration-dependent oligomerization of an alpha-helical antifreeze polypeptide makes it hyperactive. Scientific Reports, 7, 42501.

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Sedimentation Velocity Analysis of KAP1 RBCC Domain

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Sedimentation velocity data of the N-terminal RBCC domain of KAP1 (0.5 mg/ml) were obtained using a Beckman Optima XL-I analytical ultracentrifuge (Beckman Coulter) with an absorbance optical system. EPON double-sector centerpieces containing 200 μl of protein solution and 300 μl of sample buffer were centrifuged at 55,000 rpm and 20°C and data acquired with a radial increment of 0.003 cm with no delay between scans. The sedimentation of the protein was monitored at 292 and 280 nm. Data analysis was performed with sedfit (87 (link)). Buffer viscosity (0.01002 cp), density (1.04 g/cm³), and protein partial specific volumes (0.72 g/cm) were estimated using the program sednterp (88 ).

Fonti G., Marcaida M.J., Bryan L.C., Träger S., Kalantzi A.S., Helleboid P.Y., Demurtas D., Tully M.D., Grudinin S., Trono D., Fierz B, & Dal Peraro M. (2019). KAP1 is an antiparallel dimer with a functional asymmetry. Life Science Alliance, 2(4), e201900349.

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Sedimentation Velocity Analysis of IgM-FV6 Complexes

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Sedimentation velocity experiments were conducted using a Beckman Optima XL‐I analytical ultracentrifuge (Beckman Coulter) (40 000 r.p.m., 20°C). All the size exclusion chromatography‐purified protein samples were prepared in a PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄). Reference and sample were loaded into a double‐sector centrepiece and mounted in a Beckman An‐60 Ti rotor. Results from multiple scans (monitored at 280 nm) at molar ratios 1:1, 1:2 and 1:3 of IgM and FV6 (where the IgM concentration was kept constant at 0.2 μM to give an absorbance of 0.296 in a 1.2 cm path length) were fitted to a continuous size distribution using SEDFIT v.14.1 software (Schuck, 2000). Solvent density (1.00564 g ml⁻¹), viscosity (0.01020 P) and the partial‐specific volumes of IgM (0.727 cm³ g⁻¹) and FV6 (0.727 cm³ g⁻¹) were calculated using SEDNTERP software (v.20120828) (Laue et al., 1992).

Stevenson L., Huda P., Jeppesen A., Laursen E., Rowe J.A., Craig A., Streicher W., Barfod L, & Hviid L. (2015). Investigating the function of Fc‐specific binding of IgM to P lasmodium falciparum erythrocyte membrane protein 1 mediating erythrocyte rosetting. Cellular Microbiology, 17(6), 819-831.

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Oligomerization Analysis of Y BCH via AUC

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P116A (2.4 mg/ml) _YBCH were subjected to sedimentation velocity experiments using analytical ultracentrifugation to verify oligomerization. Sedimentation velocity profiles were collected by monitoring the absorbance at 280 nm. The samples were sedimented at 40,000 rpm at 24 °C for 5 h in a Beckman Optima XL-I centrifuge (Beckman Coulter Inc., Brea, CA) fitted with a four-hole AN-60 rotor and double-sector aluminium center pieces and equipped with absorbance optics. A total of 95 scans were collected and analysed using Sedfit.

Shankar S., Chew T.W., Chichili V.P., Low B.C, & Sivaraman J. (2024). Structural basis for the distinct roles of non-conserved Pro116 and conserved Tyr124 of BCH domain of yeast p50RhoGAP. Cellular and Molecular Life Sciences: CMLS, 81(1), 216.

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Characterization of Low Molecular Weight Chitosan

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LMWC was prepared by as previously described [9 (link)]. Briefly, 25 mg/mL of chitosan solution was incubated in 33% hydrogen peroxide for 3.5 hours, dialyzed against water with a molecular weight cut-off (MWCO) of 3500 Da, and freeze-dried. The molecular weight of LMWC was estimated by matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) analysis and analytical ultracentrifugation (AUC). For mass spectrometry, 1 mg/mL LMWC solution was prepared in acidified water (pH 5), filtered with a 0.2 μm syringe filter, and mixed with a matrix (sinapinic acid solution in acetonitrile/water (50:50) containing 0.1% trifluoroacetic acid) in 1:1 ratio. Mass analysis was performed with a 4800 MALDI TOF/TOF instrument (Applied Biosystems, USA) in 2000–8000 m/z range. For AUC, LMWC solution in sodium acetate buffer (pH 4.3, 10 mM) was prepared in 1, 0.5 and 0.25 mg/mL and analyzed with a Beckman Optima XL-I ultracentrifuge (Beckman Coulter Inc., CA, USA). The sedimentation coefficients and apparent molecular weights were calculated from size distribution analysis with SEDFIT v.12.0. The pH dependence of water solubility of LMWC was estimated by measuring the transmittance of LMWC solution (0.5 mg/mL) varying the pH from 2.5 to 10 with NaOH. % Transmittance (%T) was calculated as 10^−A ×100, where A was the absorbance of the solution at 500 nm.

Abouelmagd S.A., Ku Y.J, & Yeo Y. (2015). Low molecular weight chitosan-coated polymeric nanoparticles for sustained and pH-sensitive delivery of paclitaxel. Journal of drug targeting, 23(0), 725-735.

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