Bio sec 3, by Agilent Technologies - Product details

1

SAXS Characterization of A3_bGFPD Protein

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SEC-SAXS data were collected with A3_bGFPD samples at the BM29 line at the ESRF, Grenoble, with a size-exclusion HPLC column (Agilent Bio sec-3) online with a SAXS measuring cell (a 1.5 mm diameter quartz capillary in an evacuated sample chamber) and the data analysis is detailed in Note S1.
Other SAXS experiments were performed on an in-house SAXS instrument (Brüker Nanostar; λ = 1.54 Å). 30 µl of concentrated solutions (0.5 mg mL⁻¹ ≤ c ≤ 8.0 mg mL⁻¹) of A3_bGFPD were placed in a quartz capillary thermalized cell inserted into an evacuated sample chamber. SAXS data were analyzed using the program PRIMUS (https://www.embl-hamburg.de/biosaxs/primus.html).

Léger C., Di Meo T., Aumont-Nicaise M., Velours C., Durand D., Li de la Sierra-Gallay I., van Tilbeurgh H., Hildebrandt N., Desmadril M., Urvoas A., Valerio-Lepiniec M, & Minard P. (2019). Ligand-induced conformational switch in an artificial bidomain protein scaffold. Scientific Reports, 9, 1178.

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2

SAXS Analysis of Protein Structure

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SAXS data were collected at SOLEIL Light Source on beamline SWING. In line SEC-SAXS was performed using an Agilent 1200 HPLC system equipped with a 2.4 mL Bio-Sec. 3 (Agilent) column. Data were recorded on a PCCD170170 (AVIEX) detector and with a ~5–17 keV energy range allowing the collection of the angular range q between 0.0038–0.62 Å⁻¹. Samples were loaded onto the size exclusion column previously equilibrated in 25 mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM TCEP at a concentration of about 200 μM. The primary reduction of the SAXS data was performed using the software Foxtrot (http://www.synchrotron-soleil.fr/Recherche/LignesLumiere/SWING). Data processing was carried out with ATSAS (http://www.embl-hamburg.de/biosaxs/software.html) to obtain the radius of gyration (Rg), the maximum particle dimension (Dmax), the excluded particle volume (Vp) and the pair distribution function (P(r)). The program SCATTER was used to obtain the excluded particle volume (Vp). A low resolution three-dimensional ab initio model for all the samples was generated using the program DAMMIF, averaging the results of 25 independent runs using the program DAMAVER. CRYSOL was used to compare available structures with experimental scattering profiles. The ab initio models were rendered with PyMOL.

Martino L., Brown N.R., Masino L., Esposito D, & Rittinger K. (2018). Determinants of E2-ubiquitin conjugate recognition by RBR E3 ligases. Scientific Reports, 8, 68.

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3

Metalloproteome Analysis via SEC-ICP-MS

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The metalloproteomes for each condition were analyzed via SEC-ICP-MS using an Agilent Infinity II LC coupled to an Agilent 7800 ICP-MS. The methods were inspired by work done by the Roberts lab [18 (link),19 (link)]. A total of 160 µg of soluble protein (entire proteome) was injected onto an Agilent Bio-SEC 3 (3 µm, 300 Å, 4.6 × 300 mm) column and proteins were separated for 25 min using 200 mM ammonium acetate pH 8 as the mobile phase, with a flow rate of 0.4 mL/min. The five metal ion signals were monitored, and these included: ²⁴Mg, ⁵⁶Fe, ⁶³Cu, ⁶⁶Zn, and ⁷⁵As, with an integration time/mass of 1.5 s per analyte. The monitoring of ion signals was performed using the Agilent MassHunter 4.6 (version C.01.06). Metal signals were manually integrated in MassHunter, and all other data workup was performed in Microsoft Excel. Statistical differences were determined using a Student’s t test at a 95% confidence interval.

Larson J., Tokmina-Lukaszewska M., Fausset H., Spurzem S., Cox S., Cooper G., Copié V, & Bothner B. (2023). Arsenic Exposure Causes Global Changes in the Metalloproteome of Escherichia coli. Microorganisms, 11(2), 382.

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4

HPLC Analysis of Amyloid Peptides

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High performance liquid chromatography (HPLC) analysis was performed on a Perkin Elmer system that consists of a microprocessor controlled Perkin Elmer model 200 eluent delivery pump and a fixed wavelength Perkin Elmer model 200 UV-VIS spectrophotometer detection system. Samples were injected via a Perkin Elmer model 200 autosampler injector valve fitted with a 15 μl volume injector loop. The sample concentration was 10 μM. Separation was performed on a 150 mm long x 4.6 mm inner diameter column (Bio SEC-3, Agilent) with 3 μm silica absorbent. Mobile phase was isocratic 0.1 M NaCl, 0.01 M NaH₂PO₄ buffer at pH 7.4, flow rate of 0.25 ml/min, at a pressure of 34 bar (or 496 psi). Column temperature was maintained constant at 30 ^oC. All other parts of the system were maintained at room temperature (22 ± 2 ^oC). Data collection and handling were carried out by the manufacturer (Perkin Elmer) provided software. Peptide UV spectrophotometric detection was carried out at 275 nm wavelength. Seven protein standards were used to construct the calibration curve relating the retention time and the molecular weight of amyloid species (see supporting information S1 Appendix).

Pavliukeviciene B., Zentelyte A., Jankunec M., Valiuliene G., Talaikis M., Navakauskiene R., Niaura G, & Valincius G. (2019). Amyloid β oligomers inhibit growth of human cancer cells. PLoS ONE, 14(9), e0221563.

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5

Measuring Apo-PiuA-Iron Binding Kinetics

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200 μM apo-PiuA was mixed with 200 or 100 μM ⁵⁴Fe-hTf with or without 1 mM NE in NMR buffer at 35.0 °C overnight. Control incubations contained 200 μM apo-PiuA, 200 μM ⁵⁴Fe^III-NE₂-PiuA, of 200 μM ⁵⁴Fe-hTf. An Agilent 1260 Infinity II Bio-Inert LC system was used with the autosampler and column compartment cooled at 4 °C. 100 μL injections were applied to a Bio SEC-3 (Agilent) column at a flow rate of 0.35 mL/min using 200 mM NH₄NO₃, pH 7.5 as running buffer. UV profiles were monitored at 280 nm, with the flow path immediately diverted to the nebulizer of an Agilent 8800 triple quadrupole ICP-MS operating in He collision mode to detect ⁵⁴Fe counts. The ⁵⁴Fe counts were normalized to the maximum count in each spectrum.

Zhang Y., Edmonds K.A., Raines D.J., Murphy B.A., Wu H., Guo C., Nolan E.M., VanNieuwenhze M.S., Duhme-Klair A.K, & Giedroc D.P. (2020). The pneumococcal iron uptake protein A (PiuA) specifically recognizes tetradentate FeIII bis- and mono-catechol complexes. Journal of molecular biology, 432(19), 5390-5410.

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6

Recombinant expression and purification of AnAPN1

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The near full-length coding sequence of An. gambiae AnAPN1 was amplified from midgut cDNA (pMTAPN1F 5′-TACCTACCATGGCCGCCATACAAGAGTAGTGGA-3′ and pMTAPN1R 5′-GATATGGCGGCCGCCTCGGCTAGGAAGTTGGACAG-3′) and cloned into the pMT-Bip-V5-His C Drosophila expression vector using NcoI and NotI restriction enzymes. Drosophila S2 cells (Life Technologies) were stably transfected with the expression vector using Effectene Transfection Reagent (Qiagen) and grown in the presence of hygromycin B (300 μg/mL). Expression was induced upon addition of copper sulfate (600 μM), and near full-length recombinant AnAPN1 (rFL-APN1) was recovered from the supernatant 24 hours later by concentration with PEG-8000 in Spectra/Por 2 dialysis sacks (12-14 kDa molecular weight cut off) (Spectrum Labs). The protein was subsequently purified using Ni-NTA agarose (Qiagen). For further purification, 50 μl of recombinant protein was directly injected onto a Bio SEC-3 (Agilent, 150 Å, 7.8×300 mm) HPLC size-exclusion column equilibrated with 150 mM sodium phosphate, pH 7.0 (Supplementary Fig. 7), followed by buffer exchange into 10 mM Tris, 150 mM NaCl, pH 7.5, using a 3 kDa Amicon filter (EMD Millipore, Billerica, MA). Purity was assessed via SDS-PAGE and purified AnAPN1 was subsequently used in functional analyses and crystallization trials.

Atkinson S.C., Armistead J.S., Mathias D.K., Sandeu M.M., Tao D., Borhani-Dizaji N., Tarimo B.B., Morlais I., Dinglasan R.R, & Borg N.A. (2015). Structural analysis of Anopheles midgut aminopeptidase N reveals a novel malaria transmission-blocking vaccine B-cell epitope. Nature structural & molecular biology, 22(7), 532-539.

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7

SAXS Analysis of Yeast 5S RNA and Complexes

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SAXS data on S. cerevisiae free 5S RNA were collected on beamline SWING (Soleil Synchrotron) at an energy of 13 keV. SAXS data on the Rpf2–Rrs1 protein complex and the 5S–Rpf2–Rrs1 RNA–protein complex, either full-length or proteolyzed, were collected on beamline BM29 (ESRF) at an energy of 12.5 keV. Scattering data were collected at 20°C at sample concentrations between 1 and 25 mg/mL. For measurement on the 5S RNA at SWING, the sample was injected on a gel filtration column (bio-SEC 3, Agilent), and data were recorded on the in-line elution profile (David and Perez 2009 ). For proteins and protein–RNA complexes, samples were injected directly on the BM29 flow cell (Pernot et al. 2013 (link)). Buffer background scattering was collected on the gel filtration buffers used for the RNA, proteins, and RNA–protein complexes. Background subtraction, averaging, and scaling were carried out using Foxtrot on the SWING beamline or the EDNA pipeline available on the BM29 beamline.

Madru C., Lebaron S., Blaud M., Delbos L., Pipoli J., Pasmant E., Réty S, & Leulliot N. (2015). Chaperoning 5S RNA assembly. Genes & Development, 29(13), 1432-1446.

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8

SEC-SAXS Analysis of Protein Aggregation

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SEC-SAXS experiments were performed on beamline BL23A at the National Synchrotron Radiation Research Center (NSRRC, Hsinchu, Taiwan) with the capacity to separate aggregated particles on a silica-based size-exclusion column (Bio SEC-3, Agilent, United States). SAXS signals were detected by using a Pilatus detector (1M-F) and processed by an in-house developed program to obtain the SAXS profiles (Kohn et al., 2004 (link); Jeng et al., 2010 (link); Lee and Hsu, 2018 (link)). The SAXS data were collected for momentum transfer q ranging from 0.005 to 0.434 Å⁻¹, with X-ray wavelength 1.03 Å and 13 keV. The beam geometry was set to 0.5 × 0.5 mm². During the HPLC separation before SAXS measurements, the mobile phase consisted of buffer A (with and without 7 M urea) with the addition of 2% glycerol to prevent radiation damage. The protein solutions were concentrated to 10 mg/ml with the same mobile phase buffer immediately before SAXS measurements.

Hsu S.T., Lee Y.T., Mikula K.M., Backlund S.M., Tascón I., Goldman A, & Iwaï H. (2021). Tying up the Loose Ends: A Mathematically Knotted Protein. Frontiers in Chemistry, 9, 663241.

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9

SAXS Analysis of Nyx Proteins

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SAXS data were collected for NyxA and NyxB on BioSAXS beamline BM29, ESRF using an online size-exclusion chromatography setup. 50 µl of protein (10 mg ml⁻¹) were injected into a size-exclusion column (Agilent BioSec-3) equilibrated in 50 mM Tris, pH 8.0, 200 mM NaCl. Images were acquired every second for the duration of the size-exclusion run. Buffer subtraction was performed by averaging 20 frames on either side of the peak. Data reduction and analysis were performed using the BsxCuBE data collection software and the ATSAS package^{66 (link)}. The programme AutoGNOM was used to generate the pair distribution function (P(r)) and to determine D_max and R_g from the scattering curves (I(q) versus q) in an automatic, unbiased manner. Theoretical curves from the models were generated by FoXS^{67 (link)}. Ab initio modelling was performed with GASBOR^{68 (link)}.

Louche A., Blanco A., Lacerda T.L., Cancade-Veyre L., Lionnet C., Bergé C., Rolando M., Lembo F., Borg J.P., Buchrieser C., Nagahama M., Gérard F.C., Gorvel J.P., Gueguen-Chaignon V., Terradot L, & Salcedo S.P. (2023). Brucella effectors NyxA and NyxB target SENP3 to modulate the subcellular localisation of nucleolar proteins. Nature Communications, 14, 102.

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10

Recombinant expression and purification of AnAPN1

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The near full-length coding sequence of An. gambiae AnAPN1 was amplified from midgut cDNA (pMTAPN1F 5′-TACCTACCATGGCCGCCATACAAGAGTAGTGGA-3′ and pMTAPN1R 5′-GATATGGCGGCCGCCTCGGCTAGGAAGTTGGACAG-3′) and cloned into the pMT-Bip-V5-His C Drosophila expression vector using NcoI and NotI restriction enzymes. Drosophila S2 cells (Life Technologies) were stably transfected with the expression vector using Effectene Transfection Reagent (Qiagen) and grown in the presence of hygromycin B (300 μg/mL). Expression was induced upon addition of copper sulfate (600 μM), and near full-length recombinant AnAPN1 (rFL-APN1) was recovered from the supernatant 24 hours later by concentration with PEG-8000 in Spectra/Por 2 dialysis sacks (12-14 kDa molecular weight cut off) (Spectrum Labs). The protein was subsequently purified using Ni-NTA agarose (Qiagen). For further purification, 50 μl of recombinant protein was directly injected onto a Bio SEC-3 (Agilent, 150 Å, 7.8×300 mm) HPLC size-exclusion column equilibrated with 150 mM sodium phosphate, pH 7.0 (Supplementary Fig. 7), followed by buffer exchange into 10 mM Tris, 150 mM NaCl, pH 7.5, using a 3 kDa Amicon filter (EMD Millipore, Billerica, MA). Purity was assessed via SDS-PAGE and purified AnAPN1 was subsequently used in functional analyses and crystallization trials.

Atkinson S.C., Armistead J.S., Mathias D.K., Sandeu M.M., Tao D., Borhani-Dizaji N., Tarimo B.B., Morlais I., Dinglasan R.R, & Borg N.A. (2015). Structural analysis of Anopheles midgut aminopeptidase N reveals a novel malaria transmission-blocking vaccine B-cell epitope. Nature structural & molecular biology, 22(7), 532-539.

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Bio sec 3

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13 protocols using bio sec 3

SAXS Characterization of A3_bGFPD Protein

SAXS Analysis of Protein Structure

Metalloproteome Analysis via SEC-ICP-MS

HPLC Analysis of Amyloid Peptides

Measuring Apo-PiuA-Iron Binding Kinetics

Recombinant expression and purification of AnAPN1

SAXS Analysis of Yeast 5S RNA and Complexes

SEC-SAXS Analysis of Protein Aggregation

SAXS Analysis of Nyx Proteins

Recombinant expression and purification of AnAPN1

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