Superdex 200 10 300 gl column

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The Superdex 200 10/300 GL column is a size exclusion chromatography column designed for the purification and analysis of a wide range of biomolecules, including proteins, peptides, and other macromolecules. It features a prepacked, ready-to-use format with a bed volume of 24 mL and a separation range of 10,000 to 600,000 Da.

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Superdex 200 10/300 GL (342 citations) Superdex 200 10/300 (112 citations) Superdex 200 column (565 citations) Superdex 200 (746 citations)

691 protocols using superdex 200 10 300 gl column

Purification and Characterization of Malaria Proteins

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DNA corresponding to deletion constructs of PFA0660w and PfHsp70-x were PCR amplified, and cloned in pET-28a(+) bacterial expression vector before expression in E. coli BL21 (DE3) cells. Recombinant proteins were purified using Ni-NTA affinity chromatography followed by gel permeation chromatography. Gel filtration chromatography was performed using AKTA prime plus on Superdex 200 10/300 GL column (GE Healthcare Life Sciences, Chicago, IL, USA). Purified PFA0660w-S was used for raising polyclonal antibodies in rabbits commercially (Merck, Bangalore, India).
To confirm protein identity, purified recombinant protein constructs of PFA0660w and PfHsp70-x were resolved on SDS-PAGE and transferred on NC membrane. NC membrane was blocked overnight at 4 °C in 5% BSA. Following washing, the blot was probed with horseradish peroxidise (HRP) linked monoclonal anti-hexahistidine antibody (1:2000; Sigma) for 2 hours, and developed with diamino benzidine/H₂O₂ substrate (Sigma).
Recombinant ATS domain of PF08_0141 was purified for experiments as previously described^{36 ,37 (link)}. Briefly, protein was purified using Ni-NTA affinity chromatography followed by Q-Sepharose anion exchange chromatography (GE Healthcare) and then size exclusion on Superdex 200 10/300 GL column (GE Healthcare).

Behl A., Kumar V., Bisht A., Panda J.J., Hora R, & Mishra P.C. (2019). Cholesterol bound Plasmodium falciparum co-chaperone ‘PFA0660w’ complexes with major virulence factor ‘PfEMP1’ via chaperone ‘PfHsp70-x’. Scientific Reports, 9, 2664.

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Purification of Gα-1D4 Complex with PDE6

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1D4-tagged recombinant Gα_T·GTP was washed with buffer containing 20 mM Tris pH 8.0 and 5 mM MgCl₂ on a 10kD MWCO concentrator to remove DTT and then mixed with the 1D4 antibody (University of British Columbia, CA) in a 7:1 molar ratio and incubated on ice for 30 min. The mixture was loaded onto a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with buffer containing 20 mM Tris pH 8.0, and 5 mM MgCl₂ to purify the 2:1 Gα_T·GTP-1D4 complex. PDE6 was washed with buffer containing 20 mM Tris pH 8.0, and 5 mM MgCl₂ on a 100kD MWCO concentrator to remove DTT and mixed with an equal molar amount of the purified 2:1 Gα_T·GTP-1D4 complex together with 10 μM of vardenafil (Sigma-Aldrich). The mixture was incubated on ice for 30 min and purified by gel filtration chromatography using a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with buffer containing 20 mM Tris pH 8.0, 5 mM MgCl₂ and 1 μM vardenafil. Peak fractions were pooled and concentrated with a 100kD MWCO concentrator to ~10 mg/mL.

Gao Y., Eskici G., Ramachandran S., Poitevin F., Seven A.B., Panova O., Skiniotis G, & Cerione R.A. (2020). Structure of the Visual Signaling Complex between Transducin and Phosphodiesterase 6. Molecular cell, 80(2), 237-245.e4.

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Molecular Mass Analysis of LFY Proteins

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The molecular mass of GbLFY-SAM and AtLFY-SAM was estimated at 4 °C using a Superdex-200 10/300GL column (GE Healthcare), equilibrated with buffer A and calibrated with low- and high-molecular-weight protein standards (gel filtration calibration kit; GE Healthcare). Accurate molecular mass determination using SEC-MALLS was carried out with a Superdex-200 10/300GL column (GE Healthcare). GbLFY-SAM, WT and mutants were analysed in buffer A. Protein–DNA complexes containing GbLFYΔ, WT or mutants and AP1 DNA were analysed in 20 mM Tris-HCl pH 8, 150 mM NaCl, 0.25 mM EDTA, 2 mM MgCl₂ and 1 mM TCEP. Separations were performed at 20 °C with a flow rate of 0.5 ml min⁻¹. Elutions were monitored by using a DAWN-EOS detector with a laser emitting at 690 nm for online MALLS measurement (Wyatt Technology Corp., Santa Barbara, CA) and with a RI2000 detector for online refractive index measurements (Schambeck SFD). Molecular mass calculations were performed using the ASTRA software using a refractive index increment (dn/dc) of 0.185 ml g⁻¹.

Sayou C., Nanao M.H., Jamin M., Posé D., Thévenon E., Grégoire L., Tichtinsky G., Denay G., Ott F., Peirats Llobet M., Schmid M., Dumas R, & Parcy F. (2016). A SAM oligomerization domain shapes the genomic binding landscape of the LEAFY transcription factor. Nature Communications, 7, 11222.

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Characterization of Moss Protein Complexes

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Equimolar amounts of Chaetosphaeridium globosum His-tag fusion constructs were incubated in column buffer (20 mM HEPES NaOH, pH 8.0, 300 mM NaCl) and loaded onto a Superdex 200 10/300 GL column (GE Healthcare). Gel filtration runs were performed in column buffer at a flow rate of 0.4 ml/min. Selected fractions were then analyzed by SDS-PAGE and Coomassie staining. For further experiments, equimolar amounts of Physcomitrella patens His-tag fusion constructs or cpSRP RNA were mixed and incubated for 30 min at 4°C in column buffer (25 mM HEPES NaOH, pH 8.0, 300 mM NaCl, 5 mM MgCl₂, 5% (v/v) glycerol and 2 mM DTT) before loading onto a Superdex200 10/300 GL column (GE Healthcare). Gel filtration analysis was performed in column buffer at a flow rate of 0.4 ml/min and analyzed as described.

Ziehe D., Dünschede B., Zenker M., Funke S., Nowaczyk M.M, & Schünemann D. (2016). The Chloroplast SRP Systems of Chaetosphaeridium globosum and Physcomitrella patens as Intermediates in the Evolution of SRP-Dependent Protein Transport in Higher Plants. PLoS ONE, 11(11), e0166818.

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Purification of PCSK9 and LDLR-ECD

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FLAG epitope-tagged recombinant human WT PCSK9 along with A44P, R46L, R496W, and Δ31–52 variants were produced in stably transfected HEK293S cells and purified from conditioned culture medium as described previously (18 (link)). Fluorescently labeled WT PCSK9 was prepared using the DyLight800 antibody-labeling kit (Thermo Fisher Scientific) as per the manufacturer's instructions followed by gel filtration chromatography on a Superdex 200 10/300 GL column (GE Healthcare) to remove unbound dye. The extracellular domain (ECD) of LDLR used for PCSK9 binding assays was partially purified from conditioned medium of HEK293S cells cultured as described (18 (link)) stably transfected with a plasmid encoding amino acids 1–692 of human LDLR containing a C-terminal His₆ tag (a kind gift from R. Milne, University of Ottawa). LDLR-ECD was enriched by affinity chromatography using TALON superflow affinity resin (Clontech) followed by gel filtration chromatography on a Superdex 200 10/300 GL column (GE Healthcare).

Sarkar S.K., Foo A.C., Matyas A., Asikhia I., Kosenko T., Goto N.K., Vergara-Jaque A, & Lagace T.A. (2020). A transient amphipathic helix in the prodomain of PCSK9 facilitates binding to low-density lipoprotein particles. The Journal of Biological Chemistry, 295(8), 2285-2298.

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Characterizing Light-Induced PtAu1a Interactions

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Individual LOV domain containing PtAu1a variants were pre-incubated at 20°C in the dark or under continuous blue light illumination (400 µW cm^–2 at 450 nm) from a royal blue (455 nm) collimated LED lamp (Thorlabs) for 20 min. 100 µl of a 150 µM protein solution was subjected to size-exclusion chromatography at RT on a Superdex 200 10/300 GL column (GE Healthcare) equilibrated in buffer C. For dark and light experiments, the column was kept in the dark or continuously illuminated with blue light during the gel-filtration runs. To investigate the interaction between PtAu1a_LOV and PtAu1a_bZIP, the proteins were mixed in a molar ratio of 1:1 and concentrated using centrifugal filter units (Amicon, 3 kDa cut off). 100 µl of a 75 µM complex solution (based on a theoretical (PtAu1a_LOV-PtAu1a_bZIP)2 complex) was subjected to size-exclusion chromatography at RT on a Superdex 200 10/300 GL column (GE Healthcare) equilibrated in buffer C. The high performance liquid chromatography (HPLC) (Waters) setup was connected to a MALS detector (Dawn Heleos, Wyatt Technology) combined with a refractive-index detector (Waters). Data analysis was performed using the ASTRA software (Wyatt Technology), providing estimates for the molar mass of the different PtAu1a variants and the (PtAu1a_LOV-PtAu1a_bZIP)₂ complex.

Heintz U, & Schlichting I. (2016). Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence. eLife, 5, e11860.

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Oligomeric States and Interactions of Vps4 Mutants

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To examine the oligomeric states of Vps4 mutants, each mutant protein at 100 μM was incubated with 1 mM ATP at 4 °C for 1 h, and then loaded onto a Superdex 200 10/300 GL column (GE Healthcare) preequilibrated with the assembly buffer. To examine the interactions between Vps4 and Vta1 in the absence of ATP, Vps4 mutants at 100 μM were incubated with Vta1 at a molar ratio of 1:1.5 in the absence of ATP at 4 °C for 1 h before being loaded onto the Superdex 200 10/300 GL column (GE Healthcare) preequilibrated with the FPLC buffer. To examine the interactions between Vps4 and Vta1 in the presence of ATP, Vps4 mutants at 5 μM were incubated with Vta1 at a molar ratio of 1:2 in the presence of 1 mM ATP at 4 °C for 1 hour before being loaded onto the Superose 6 10/300 GL column (GE Healthcare) preequilibrated with the assembly buffer.

Sun S., Li L., Yang F., Wang X., Fan F., Yang M., Chen C., Li X., Wang H.W, & Sui S.F. (2017). Cryo-EM structures of the ATP-bound Vps4E233Q hexamer and its complex with Vta1 at near-atomic resolution. Nature Communications, 8, 16064.

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Structural Characterization of SARS-CoV-2 Variants

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Purified hACE2 was mixed and incubated with BF.7 RBD, XBB RBD, XBB.1.5 RBD, BQ.1 RBD and BQ.1.1 RBD at a 1:1.5 molar ratio (hACE2 to RBD) on ice for about 2 h. The six mixtures were then purified on Superdex™ 200 10/300 GL column (GE Healthcare) in a buffer containing 20 mM Tris (pH 8.0) and 150 mM NaCl. The XBB.1.5 RBD/hACE2 was mixed with S304 Fab at a 1:1.5 molar ratio (hACE2-RBD to Fab) on ice for about 2 h and purified on Superdex™ 200 10/300 GL column (GE Healthcare) with 20 mM Tris (pH 8.0) and 150 mM NaCl. Purified complex proteins (BF.7 RBD/hACE2, XBB RBD/hACE2, XBB.1.5 RBD/hACE2/S304 Fab, BQ.1 RBD/hACE2 and BQ.1.1 RBD/hACE2) were concentrated to 2 mg/mL for cryo-EM using a 30-kDa cut-off Ultracon concentrator (Millipore).

Li W., Xu Z., Niu T., Xie Y., Zhao Z., Li D., He Q., Sun W., Shi K., Guo W., Chang Z., Liu K., Fan Z., Qi J, & Gao G.F. (2024). Key mechanistic features of the trade-off between antibody escape and host cell binding in the SARS-CoV-2 Omicron variant spike proteins. The EMBO Journal, 43(8), 1484-1498.

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Structural Characterization of Frizzled-G Protein Complexes

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About the complex formation of Frizzled-G protein. Purified Frizzled receptor, heterodimeric Gβ1γ2, miniGαs (or miniGαq), and Nb35 were mixed in a 1:1.2:1.5:2 molar ratio followed by the addition of apyrase (1 unit), respectively. The mixture was incubated at 4 °C overnight. The FZD1-Gq (FZD3-Gs or FZD6-Gs) complex was loaded on size-exclusion chromatography (Superdex 200 10/300 GL column, GE) in SEC buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 0.00075% (w/v) LMNG, 0.00025% (w/v) glyco-diosgenin (GDN, Anatrace), 0.00025% (w/v) CHS, and 100 µM DTT). Peak fractions containing FZD-G protein complex were concentrated to 2.5 mg/mL for electron microscopy studies.
About the complex formation of inactive Frizzled protein. Purified Frizzled receptor, anti-BRIL Fab and VHH were mixed in a 1:1.5:2 molar ratio, respectively. The mixture was incubated at 4 °C overnight. The FZD1-Fab-VHH (FZD3-Fab-VHH or FZD6-Fab-VHH) complex was loaded on size-exclusion chromatography (Superdex 200 10/300 GL column, GE) in SEC buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 0.00075% (w/v) LMNG, 0.00025% (w/v) GDN, 0.00025% (w/v) CHS, and 100 µM DTT). Peak fractions containing complex were concentrated to 2.5 mg/mL for electron microscopy studies.

Zhang Z., Lin X., Wei L., Wu Y., Xu L., Wu L., Wei X., Zhao S., Zhu X, & Xu F. (2024). A framework for Frizzled-G protein coupling and implications to the PCP signaling pathways. Cell Discovery, 10, 3.

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Purification of Recombinant Puf6 Protein

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Recombinant His6-Puf6 protein was expressed in E. coli BL21 pRARE cells (Merck Millipore, Darmstadt, Germany) at 20 °C for 18 h and 0.5 mM IPTG. His6-tagged proteins were affinity purified by gravity flow batch purification using cOmplete His-Tag purification Resin (Roche AG, Basel, Switzerland) in 500 mM NaCl, 20 mM HEPES pH 7.5, 5 mM MgCl₂, 5 mM β-mercaptoethanol, 1 mM TCEP. Eluates were concentrated and further purified through size exclusion chromatography in 150 mM NaCl, 20 mM HEPES pH7.5, 5 mM MgCl₂, 5 mM β-mercaptoethanol on a Superdex 200 10/300 GL column (GE Healthcare). For ensemble FRET, His6-GB1-Puf6-PUM (161-656aa) was expressed and purified as described above in 500 mM KCl, 20 mM HEPES pH 7.4, 1 mM DTT, 1 mM TCEP, 0.05% Triton-X. Eluates were concentrated, dialyzed into 100 mM KCl, 20 mM HEPES pH 7.5, 1 mM DTT, and further purified through size exclusion chromatography in 100 mM KCl, 20 mM HEPES pH7.5, 1 mM DTT on a Superdex 200 10/300 GL column (GE Healthcare).

Gerhardy S., Oborská-Oplová M., Gillet L., Börner R., van Nues R., Leitner A., Michel E., Petkowski J.J., Granneman S., Sigel R.K., Aebersold R, & Panse V.G. (2021). Puf6 primes 60S pre-ribosome nuclear export at low temperature. Nature Communications, 12, 4696.

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