All proteins were purified as described above. Putative complexes were reconstituted prior to analytical gel filtration by mixing stoichiometric molar ratios of Ctf4CTD with either GINSPsf1ΔC or GINSPsf1ΔC,Sld5ΔN followed by centrifugation at 16,000g for 10 minutes at 4°C to remove potential aggregates. 100 μl samples of Ctf4CTD, GINSPsf1ΔC, GINSPsf1ΔC,Sld5ΔN, Ctf4CTD-GINSPsf1ΔC and Ctf4CTD-GINSPsf1ΔC,Sld5ΔN were subsequently fractionated over a Superdex S200 HR 10/300 column (GE Healthcare) preequilibrated in 20 mM HEPES pH 7.2, 140 mM KCl.
Superdex s200 hr 10 300 column
Manufactured by GE Healthcare
Sourced in Sweden
The Superdex S200 HR 10/300 column is a size exclusion chromatography column designed for the separation and analysis of proteins, peptides, and other macromolecules. The column features a Superdex matrix and a bed volume of 24 mL, with a separation range of 10,000 to 600,000 daltons. The column dimensions are 10 mm in diameter and 300 mm in length.
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Lab products found in correlation
5 protocols using superdex s200 hr 10 300 column
1
Reconstitution of Ctf4-GINS Complexes
2
Purification and Size Determination of Mutant Proteins
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Gel filtration chromatography was performed in a buffer comprising 10 mM HEPES (pH 8.0), 100 mM NaCl referred to as buffer-B hereafter. Highly concentrated (∼20 mg/ml) purified mutant proteins (S276C and S306A) were separately applied to a Superdex S200 HR 10/300 column (GE Healthcare, Uppsala, Sweden) that was pre-equilibrated in buffer-B. Proteins were then eluted in the same buffer at a flow rate of 0.2 ml/min. The standards used for calibration were as follows: BSA 66 kDa, alcohol dehydrogenase 150 kDa, and β-amylase 200 kDa. Elution volume (Ve)/void volume (V0) compared with log of molecular masses of standards was plotted to generate the calibration curve from which MWs of hHtrA2 and its variants were calculated as described earlier [20 (link)].
3
Reconstitution of Ctf4-GINS Complexes
4
Protein Complex Formation Evaluation
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Physical interaction between the E1-like and E2-like proteins was examined by size-exclusion chromatography using an analytical Superdex S200 HR 10/300 column (GE Healthcare). The E1/E2 complex was formed at 60 °C, before the gel filtration analysis by mixing together 250 μg of each protein in a final volume of 500 μl gel filtration buffer (20 mM Tris [pH 8.0], 300 mM NaCl, 5% glycerol, 1 mM DTT). Reactions were subsequently spun at 16,000 g in a benchtop centrifuge for 5 min to remove any precipitated material, before loading onto the size exclusion chromatography column. 0.5 ml fractions were collected and resolved by SDS-PAGE, on 15% polyacrylamide gels. The proteins were visualised with Coomassie stain.
5
Size-Exclusion Chromatography of ESCRT-I Complexes
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Heimdallarchaeota Vps22 (27.9 kDa) was subjected to analytical SEC using a Superdex 200 16/600 size-exclusion column (GE Healthcare). The sample was loaded onto the column in a buffer comprised of 20 mM Tris-HCl pH 8.0, 200 mM NaCl and 5% (v/v) glycerol at a flow rate of 0.5 mL/min. The calibration curve was established under the same conditions using the following standard proteins (Sigma MWGF1000): carbonic anhydrase (CAN; 29 kDa), bovine serum albumin (BSA; 66 kDa), alcohol dehydrogenase (ADH; 150 kDa), beta-amylase (BAM; 200 kDa), apoferritin (AFE; 443 kDa) and thyroglobulin (TGL; 669 kDa). Physical interactions between the Odinarchaeota ESCRT-I complex proteins (Vps23, Vps28 and ubiquitin) were examined by size-exclusion chromatography using an analytical Superdex S200 HR 10/300 column (GE Healthcare). Prior to the gel filtration analyses, ESCRT-I complexes were formed at 60 °C by mixing 250 μg of each protein in a final volume of 500 μl gel filtration buffer (20 mM Tris [pH 8.0], 150 mM NaCl, 5% glycerol, 1 mM DTT) for 10 min. The complexes were subsequently spun at 16,000 g in a benchtop centrifuge for 5 min to remove any precipitated material, before loading onto the size-exclusion chromatography column. 0.5 ml fractions were collected and resolved by SDS-PAGE, on 15% polyacrylamide gels. The proteins were then visualized with Coomassie stain.
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