LRX–RALF complexes were obtained in vivo by coexpression in insect cells. The apo proteins were obtained by complex dissociation in vitro using a low pH buffer. After purification of the LRX–RALF complex (see above), the protein complex was incubated for 1 h at 4 °C in the following buffer: 20 mM citric acid pH 2, 150 mM NaCl. Next, the dissociated proteins were separated by gel filtration, using a Superdex 200 increase 10/300 GL column (GE Healthcare) equilibrated in the same buffer. Each protein was then collected separately and dialyzed against 20 mM citric acid pH 5, 150 mM NaCl. To test whether proteins were still folded and functional, proteins were again run in a Superdex 200 increase 10/300 GL column (GE Healthcare) in 20 mM citric acid pH 5, 150 mM NaCl. We also tested, for each batch of dissociated protein, that the complex would be fully reconstituted when mixing the proteins in equimolar proportions. Additionally, we tested the in vivo activity in pollen tube growth assays of the dissociated peptide (folded) versus a synthetic linear version of RALF4 of the same sequence.
Superdex 200 increase 10 300 gl column
Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom, Germany, China, Japan
The Superdex 200 Increase 10/300 GL column is a size exclusion chromatography column designed for the separation and purification of proteins, peptides, and other biomolecules. The column has a bed volume of 24 mL and is compatible with a wide range of sample sizes and flow rates.
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Lab products found in correlation
Astra 6, by Wyatt Technology (23 mentions)
Kta pure system, by GE Healthcare (14 mentions)
Optilab t rex, by Wyatt Technology (13 mentions)
Histrap hp column, by GE Healthcare (12 mentions)
Minidawn treos, by Wyatt Technology (11 mentions)
Gel filtration standard, by Bio-Rad (10 mentions)
Minidawn treos detector, by Wyatt Technology (9 mentions)
Hispur ni nta resin, by Thermo Fisher Scientific (9 mentions)
Astra software, by Wyatt Technology (9 mentions)
Expi293f cells, by Thermo Fisher Scientific (9 mentions)
Superose 6 increase 10 300 gl column, by GE Healthcare (8 mentions)
Dawn heleos 2, by Wyatt Technology (8 mentions)
Akta fplc system, by GE Healthcare (7 mentions)
Astra 7, by Wyatt Technology (7 mentions)
Optilab t rex refractometer, by Wyatt Technology (6 mentions)
Optilab t rex refractive index detector, by Wyatt Technology (6 mentions)
Astra v 6, by Wyatt Technology (6 mentions)
Opti mem, by Thermo Fisher Scientific (6 mentions)
Akta purifier, by GE Healthcare (6 mentions)
Rid 20a refractive index detector, by Shimadzu (5 mentions)
576 protocols using superdex 200 increase 10 300 gl column
1
Dissociation and Reconstitution of LRX-RALF Complexes
2
Superdex 200 Size Exclusion Chromatography
3
Structural analysis of PorX_Fj by SEC-MALS
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For the Size-Exclusion Chromatography Multi-Angle Light Scattering (SEC-MALS) analysis, the purified PorXFj (1.6 mg mL−1) was incubated for 1 h at RT with 20 mM acetyl phosphate (AcP) + 10 mM MgCl2, or 20 mM AcP, or 10 mM MgCl2, or 100 µM ZnCl2. The samples were loaded on a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated in PBS at a flow rate of 0.6 mL min−1, using an Ultimate 3000 HPLC system (Fischer Scientific). Detection was performed using an eight-angle light-scattering detector (DAWN8, Wyatt Technology) and a differential refractometer (Optilab, Wyatt Technology).
For the Size-Exclusion Chromatography (SEC) analysis, the purified PorXFj (1.6 mg mL−1) was incubated for 1 h at RT with 100 µM ZnCl2, MnCl2, or CuCl2. The samples were loaded on a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated in PBS supplemented with100 µM of the corresponding metal solution.
For the Size-Exclusion Chromatography (SEC) analysis, the purified PorXFj (1.6 mg mL−1) was incubated for 1 h at RT with 100 µM ZnCl2, MnCl2, or CuCl2. The samples were loaded on a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated in PBS supplemented with100 µM of the corresponding metal solution.
4
Purification and Analysis of sNASP TPR Domain
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The sNASP TPR domain was loaded onto a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated in a buffer containing 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 5% glycerol. Peak 1 contained fraction eluted between 11.5 and 13.0 mL, and peak 2 contained fractions eluted between 14.0 and 15.5 mL. Samples of both peak 1 and peak 2 were reloaded onto the sizing column for further analysis. Analyses of various protein complexes and individual components were carried out using the samples prepared as described before, and in the cases of testing binding with sNASP TPR domain, other components were added in roughly twofold excess. The loading samples were generally diluted to ∼1–3 mg/mL, and a total volume of 0.5 mL in 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 5% glycerol. The samples were then injected at 0.4 mL/min onto a Superdex 200 Increase 10/300 GL column (GE Healthcare). Elution fractions between 8.0 and 19.5 mL were pooled and analyzed by SDS-PAGE and stained with Coomassie blue. The gel filtration chromatograms were plotted by OriginPro 8.0 (OriginLab Corporation).
5
Isolating B cell-binding E2 protein
6
Cryo-EM of Phosphorylated NTSR1-βarr1 Complex
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For cryo-EM, phosphorylated NTSR1 was mixed in an equimolar ratio of βarr1ΔCT at a concentration of ~5 μM, supplemented with NTS8–13 to 10 μM final and incubated at 25 °C for 30 minutes before being concentrated with a 50 kDa MWCO concentrator (Amicon or Vivaspin) to ~350 μL and purified by size-exclusion chromatography using two tandem Superdex 200 increase 10/300 GL columns (GE healthcare). The mobile phase used was 20 mM HEPES pH 7.4, 100 mM NaCl, 0.00075% LMNG, 0.00025% GDN 0.0001% CHS and 0.2 μM NTS8–13. Fractions containing complex were combined and diluted to 1 μM final and sulfo-LC-SDA (sulfo-NHS-LC-Diazirine) (sulfosuccinimidyl 6-(4,4’-azipentanamido)hexanoate) (Thermo Fisher), as a solution in DMSO, was added to 250 μM final and such that the final DMSO concentration was below 4%. The reaction was allowed to proceed for 45 minutes at 25°C in the dark, before hydroxylamine was added to a final concentration of 3 mM and incubated for an additional 15 minutes. The sample was distributed into a clear 96-well plate (90 μL/well), put on ice and irradiated for 45 minutes using a UVL-56 lamp. The sample was then pooled and concentrated to ~500 μL then re-run by SEC, again using two tandem Superdex 200 increase 10/300 GL columns (GE healthcare). Peak fractions were combined and concentrated with a 100 kDa MWCO concentrator (Amicon) to a final concentration of 4.5 mg/mL.
7
Cryo-EM of Phosphorylated NTSR1-βarr1 Complex
8
Purification of Recombinant Proteins
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The proteins were expressed in HEK293F cells. Cell culture supernatants were collected after 5 days of transfection. Cell culture supernatants were collected, filtered through 0.22 µm filters, and purified by His-Trap HP (GE Healthcare) and SuperdexTM 200 Increase 10/300 Gl (GE Healthcare) size exclusion chromatography. The purified proteins were stored in a protein buffer (20 mM Tris-HCl, 150 mM NaCl, pH 8.0).
For the rabbit ACE2 used in structure determination, the plasmids were transformed into E. coli BL21 (DE3) cells and overexpressed as inclusion bodies under 1 mM IPTG induction. The inclusion bodies were then dissolved by dissolution buffer [6 M Gua-HCl, 10% vol/vol glycerol, 50 mM Tris-HCl, 100 mM NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0] and refolded. Briefly, 5 mL of the solution (30 mg/mL) was added drop by drop to 2.5 L refolding buffer [100 mM Tris-HCl, 400 mM L-Arg-HCl, 2 mM EDTA, 5 mM glutathione (GSH), and 0.5 mM oxidized glutathione (GSSG), pH 8.0]. After gently stirring for 8 h, we concentrated and exchanged proteins into a buffer containing 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl and purified using a SuperdexTM 200 Increase 10/300 Gl column (GE Healthcare).
For the rabbit ACE2 used in structure determination, the plasmids were transformed into E. coli BL21 (DE3) cells and overexpressed as inclusion bodies under 1 mM IPTG induction. The inclusion bodies were then dissolved by dissolution buffer [6 M Gua-HCl, 10% vol/vol glycerol, 50 mM Tris-HCl, 100 mM NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0] and refolded. Briefly, 5 mL of the solution (30 mg/mL) was added drop by drop to 2.5 L refolding buffer [100 mM Tris-HCl, 400 mM L-Arg-HCl, 2 mM EDTA, 5 mM glutathione (GSH), and 0.5 mM oxidized glutathione (GSSG), pH 8.0]. After gently stirring for 8 h, we concentrated and exchanged proteins into a buffer containing 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl and purified using a SuperdexTM 200 Increase 10/300 Gl column (GE Healthcare).
9
Expression and Purification of hACE2-mFc and SARS-CoV-2 RBDs
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The hACE2 fused with mFc were expressed and purified from the culture supernatants of HEK293F cells using a Protein A affinity column (GE Healthcare) and further purified by gel filtration using a SuperdexTM 200 10/300 GL (GE Healthcare). Purified proteins were stored in a buffer containing 20 mM Tris-HCl and 150 mM NaCl (pH 8.0). Proteins for SPR assay were transferred to PBST (1.8 mM KH2PO4, 10 mM Na2HPO4 (pH 7.4), 137 mM NaCl, 2.7 mM KCl, and 0.005% (v/v) Tween 20) buffer.
The hACE2 and RBDs from original SARS-CoV-2 and VOCs cloned in pCAGGS were expressed in HEK293F cells. Cell culture supernatants were collected, filtered with a 0.22 μm filter, purified by His-Trap HP column (GE Healthcare), and SuperdexTM 200 Increase 10/300 GL column (GE Healthcare). Purified proteins were stored in protein buffer (20 mM Tris-HCl, pH 8.0 and 150 mM NaCl).
The hACE2 and RBDs from original SARS-CoV-2 and VOCs cloned in pCAGGS were expressed in HEK293F cells. Cell culture supernatants were collected, filtered with a 0.22 μm filter, purified by His-Trap HP column (GE Healthcare), and SuperdexTM 200 Increase 10/300 GL column (GE Healthcare). Purified proteins were stored in protein buffer (20 mM Tris-HCl, pH 8.0 and 150 mM NaCl).
10
Expression and Purification of scFv16
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The expression and purification of scFv16 were performed as previously described39 (link). In brief, scFv16 secretion in the supernatant of transfected High Five cells was collected by affinity chromatography using Ni-Sepharose columns. The eluate was purified by size-exclusion chromatography with a SuperdexTM 200 Increase 10/300 GL column (GE Healthcare). Then, the monomeric fractions were concentrated, flash frozen, and stored at −80 °C until use.
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