Superdex 200 10 300 gl

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The Superdex 200 10/300 GL is a pre-packed size exclusion chromatography column designed for high-resolution fractionation of proteins, peptides, and other biomolecules. The column is made of a durable borosilicate glass column and filled with a Superdex 200 resin. It has a bed volume of 24 ml and a recommended flow rate range of 0.2-0.8 ml/min. The Superdex 200 resin provides effective separation of molecules with molecular weights between 10,000 and 600,000 daltons.

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342 protocols using superdex 200 10 300 gl

Structural Analysis of Omicron Spike Complexes

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For the ACE2-bound or S309-bound Omicron spike and ACE2-bound Omicron spike with L371S, P373S, and F375S mutations, the spike protein was incubated with the purified hACE2 at a 1:4 molar ratio (spike trimer to hACE2) overnight at 4 °C before purification by concentration and dialysis using a 100-kDa cut-off Ultracon concentrator (Millipore) with 20 mM Tris (pH 8.0) and 150 mM NaCl. For the Omicron RBD-hACE2-S304 complex, RBD and hACE2 were co-incubated for 2 h at 4 °C at a 1:1.2 molar ratio. The mixture was then purified via gel filtration using a Superdex^TM 200 10/300 GL (GE Healthcare) to obtain the Omicron RRD-hACE2 complex. The Omicron RBD-hACE2 and S304 Fab were then mixed at a 1:1.5 molar ratio and incubated overnight at 4 °C, followed by gel filtration using a Superdex^TM 200 10/300 GL (GE Healthcare) to obtain Omicron RBD-hACE2-S304 complex. The protein complex was stored in a buffer containing 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl for cryo-EM sample preparation.

Zhao Z., Zhou J., Tian M., Huang M., Liu S., Xie Y., Han P., Bai C., Han P., Zheng A., Fu L., Gao Y., Peng Q., Li Y., Chai Y., Zhang Z., Zhao X., Song H., Qi J., Wang Q., Wang P, & Gao G.F. (2022). Omicron SARS-CoV-2 mutations stabilize spike up-RBD conformation and lead to a non-RBM-binding monoclonal antibody escape. Nature Communications, 13, 4958.

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SEC-MALLS Analysis of USP12-UAF1 Complexes

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The SEC-MALLS experiments were performed using the miniDawn Tristar light scattering detector (Wyatt technologies, USA) in line with size exclusion chromatography. After equilibration of the Superdex 200 10/300 GL and the Superose 6 increase 10/300 GL (GE, USA) in buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM DTT, the USP12_FL-Ub/UAF1₅₈₀ (8 μM) and the USP1/UAF1_FL complex (6 μM) were loaded on the Superdex 200 10/300 GL while the USP12_FL-Ub/UAF1_FL sample (42 μM) was loaded on the Superose 6 increase 10/300 GL (GE, USA). Molecular weight estimation was done by using the refractive index signal as measure of the concentration with the Astra software (Wyatt Technologies, USA).

Dharadhar S., Clerici M., van Dijk W.J., Fish A, & Sixma T.K. (2016). A conserved two-step binding for the UAF1 regulator to the USP12 deubiquitinating enzyme. Journal of Structural Biology, 196(3), 437-447.

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Protein Assembly Chromatography

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For unassembled samples, 10 μM M1 in 50 mM HEPES (pH 7) were loaded onto a Superdex 200 10/300 GL (GE Healthcare Life Sciences) equilibrated in 50 mM HEPES (pH 7) at 4°C. To test for assembly, 10 μM of M1 and 10–80 μM of NTD^M1 were assembled by the addition of cold 2 M NaCl and loaded onto a Superdex 200 10/300 GL (GE Healthcare Life Sciences) equilibrated in 50 mM HEPES (pH 7), 2 M NaCl at 4°C.

Selzer L., Su Z., Pintilie G.D., Chiu W, & Kirkegaard K. (2020). Full-length three-dimensional structure of the influenza A virus M1 protein and its organization into a matrix layer. PLoS Biology, 18(9), e3000827.

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Isolation of Snake Venom Metalloproteinases

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The PI SVMP BaP1 was isolated from the venom of Bothrops asper, as described by Gutiérrez et al. [22 (link)] and Watanabe et al. [23 (link)], by a combination of ion-exchange chromatography on CM-Sepharose, followed by affinity chromatography on Affi-gel Blue. The PIII SVMP CsH1 was isolated from Crotalus simus venom, as described by Herrera et al. [7 (link)], by ion-exchange chromatography on DEAE-Sepharose, followed by gel filtration on a Superdex TM 200 10/300GL (GE Healthcare, LifeSciences) column (10 x 300 mm) using an ÄKTA FPLC (GE Healthcare, Life Sciences). Homogeneity of SVMP preparations was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Both toxins were isolated from the venoms of more than 40 adult specimens of each species collected in Costa Rica and maintained at the serpentarium of Instituto Clodomiro Picado, Costa Rica. After collection, venoms of each species were separately pooled, lyophilized, and stored at -20°C until used. The Minimum Hemorrhagic Dose (MHD), corresponding to the amount of enzyme that induces a hemorrhagic spot of 10 mm diameter in mice 2 h after injection, is 20 μg for BaP1 [22 (link)] and 2.2 μg for CsH1 [7 (link)].

Herrera C., Voisin M.B., Escalante T., Rucavado A., Nourshargh S, & Gutiérrez J.M. (2016). Effects of PI and PIII Snake Venom Haemorrhagic Metalloproteinases on the Microvasculature: A Confocal Microscopy Study on the Mouse Cremaster Muscle. PLoS ONE, 11(12), e0168643.

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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).

Han P., Li L., Liu S., Wang Q., Zhang D., Xu Z., Han P., Li X., Peng Q., Su C., Huang B., Li D., Zhang R., Tian M., Fu L., Gao Y., Zhao X., Liu K., Qi J., Gao G.F, & Wang P. (2022). Receptor binding and complex structures of human ACE2 to spike RBD from omicron and delta SARS-CoV-2. Cell, 185(4), 630-640.e10.

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Purified PICP Characterization by FPLC

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The purified PICP was analyzed in the Pharmacia AKTA FPLC system using a Superdex^TM 200 10/300 GL (10 × 300 mm; GE Healthcare) size exclusion column. After 1 mL injection of the purified PICP (2 mg/mL), the protein was eluted with TBS buffer (500 mM NaCl, 2.7 mM KCl, 12 mM Tris, pH 7.4) at a flow rate 0.35 mL/min. The purified PIIICP was analyzed in the Agilent 1160 HPLC system using the Superdex^TM 200 10/300 GL column (GE Healthcare). After 50 µL injection of the purified PICP (2 mg/mL), the protein was eluted with PBS buffer (pH 7.4) at a flow rate 0.75 mL/min. Chromatograms were obtained by monitoring the absorbance at 280 nm. To determine the apparent molecular mass of the protein, standard molecular weight markers (Blue dextran, 3000 kDa; alcohol dehydrogenase, 150 kDa; bovine serum albumin, 66 kDa; cytochrome c, 12 kDa; Sigma-Aldrich) were used.

Seo W.Y., Kim J.H., Baek D.S., Kim S.J., Kang S., Yang W.S., Song J.A., Lee M.S., Kim S, & Kim Y.S. (2017). Production of recombinant human procollagen type I C-terminal propeptide and establishment of a sandwich ELISA for quantification. Scientific Reports, 7, 15946.

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Dimerization analysis of sulfatase mutants

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To analyze if the N-terminal Cys residue contributed to the dimerization of the sulfatase, the mutants of endoVB4SF-C27A and corresponding exoPB4SF-Q5C were produced using a Fast Mutagenesis System Kit (TransGen) as previously described by Prabhakar et al. (2005) (link) The plasmids were amplified using the primer pairs 5′-ATGGTGACTGCTGGCGCGTCGGCTAC-3′ (forward, endoVB4SF-C27A) and 5′-CGCGCCAGCAGTCACC ATGGTTGC-3′ (reverse, endoVB4SF-C27A), and 5′-TTCTTGA CGGGCAATTGCCCCGCTG-3′ (forward, exoPB4SF-Q5C) and 5′-GCAATTGCCCGTCAAGAATTCGGATCCG-3′ (reverse, exoPB4SF-Q5C). The plasmids of the mutants were amplified by transferring them to DMT Chemically Competent Cells (TransGen), and mutated clones were confirmed by DNA sequencing at Sangon Biotech Co., Ltd. (Shanghai, China). Next, the correct mutants were transferred into E. coli BL21 (DE3) cells. endoVB4SF-C27A and exoPB4SF-Q5C were expressed and purified as described above for the wild-type enzyme.
The purified endoVB4SF, exoPB4SF, endoVB4SF-C27A and exoPB4SF-Q5C proteins were detected at 280 nm through size-exclusion chromatography (Superdex^TM 200 10/300 GL, GE Healthcare) eluted with buffer (10 mM Tris-HCl and 100 mM NaCl, pH 8.0) with or without 5 mM DTT.

Wang S., Su T., Zhang Q., Guan J., He J., Gu L, & Li F. (2019). Comparative Study of Two Chondroitin Sulfate/Dermatan Sulfate 4-O-Sulfatases With High Identity. Frontiers in Microbiology, 10, 1309.

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Preparation and Characterization of MILL Protein Tetramers

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cDNAs for MILL1 and MILL2 mutants without stop codon were generated by PCR. The primer sequences that incorporated PshBI and BamHI sites to facilitate cloning are shown in Supplementary Table 3. The PCR products were digested with PshBI/BamHI and ligated to the NdeI/BamHI-digested pGMT7-birA vector^{59 (link)}. Recombinant soluble MILL1-birA/β₂m and MILL2-birA/β₂m heterodimers incorporating the C-terminal biotin ligase (BirA) recognition sequence (GSLHHILDAQKMVWNHR) were prepared by the same procedure mentioned above. Refolded MILL1-birA/β₂m and MILL2-birA/β₂m were biotinylated with BirA enzyme (Avidity). After biotinylation, biotinylated MILLs were separated from free biotin by size-exclusion chromatography with Superdex^TM 200 10/300 GL (GE Healthcare). MILL tetramer was produced by incubating biotinylated MILL/β₂m with phycoerythrin (PE)-conjugated streptavidin (Thermo) for 15 min on ice. For surface staining with MILL tetramer, NIH-3T3 cells (RCB0150, RIKEN Cell Bank) were detached by DPBS(-) supplemented with 5 mM EDTA, and cell suspensions were stained with PE-conjugated MILL tetramer on ice for 15 min. Stained cells were washed with DPBS(-) supplemented with 2% FBS and binding of MILL tetramers was analyzed by flow cytometry.

Kajikawa M., Ose T., Fukunaga Y., Okabe Y., Matsumoto N., Yonezawa K., Shimizu N., Kollnberger S., Kasahara M, & Maenaka K. (2018). Structure of MHC class I-like MILL2 reveals heparan-sulfate binding and interdomain flexibility. Nature Communications, 9, 4330.

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Fucoidan from Kjellmaniella crassifolia Purification

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Unless specified, all chemicals are of analytical grade and were purchased from Sigma (St. Louis, DE, USA) or Aladdin (Shanghai, China). Escherichia coli Trans10 and BL21 (DE3) were purchased from TransGen Biotech (Beijing, China). Crystallization screen commercial kits were purchased from Hampton Research (Aliso Viejo, CA, USA). Ni²⁺-NTA column, Superdex^TM 200 10/300 GL, and DEAE Sepharose Fast Flow were purchased from GE Life Sciences and GE Healthcare ((Chicago, IL, USA). Fucoidan from Kjellmaniella crassifolia used in this experiment was given by Professor Qiukuan Wang of Dalian Ocean University (Dalian, China).

Wang J., Liu Z., Pan X., Wang N., Li L., Du Y., Li J, & Li M. (2022). Structural and Biochemical Analysis Reveals Catalytic Mechanism of Fucoidan Lyase from Flavobacterium sp. SA-0082. Marine Drugs, 20(8), 533.

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Preparation and Labeling of TM-MBP

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OmpF and BtuB were purified according to previously published protocols10 (link),12 (link). The purified proteins contained some endogenous LPS carried through the purification.An artificial transmembrane domain (ALAALAALAALAALAALAALAKSSR) was fused to the C-terminus of maltose-binding protein (TM-MBP) by insertion of the corresponding oligonucleotide linker into the MCS of pMALc2x via restriction with BamHI and HindIII34 (link). For efficient purification and fluorescent labelling, an N-terminal His₁₀-tag and a ybbR-tag (DSLEFIASKLA)35 (link) were inserted via the NdeI site. This protein was expressed in E. coli TG-1 cells at 37 C according to standard protocols. After cell lysis, the membrane fraction was solubilized with buffer containing 20 mM Triton X-100 and the protein purified by immobilized metal ion affinity chromatography (5 ml HiTrap Chelating, GE Healthcare). The protein was then covalently modified using a 2-fold molar excess of Coenzyme A-Dy647 in the presence of 5 µM of the phosphopantetheinyl transferase Sfp and 10 mM Mg²⁺ for 1 h at room temperature35 (link). Finally, labelled TM-MBP (TM-MBP^Dy647) was further purified by size exclusion chromatography in 20 mM Hepes, 150 mM NaCl pH 7.5 containing 0.6 mM Triton X-100 (Superdex 200 10/300 GL, GE Healthcare).

Rassam P., Copeland N.A., Birkholz O., Tóth C., Chavent M., Duncan A.L., Cross S.J., Housden N.G., Kaminska R., Seger U., Quinn D.M., Garrod T.J., Sansom M.S., Piehler J., Baumann C.G, & Kleanthous C. (2015). Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria. Nature, 523(7560), 333-336.

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