For advanced purification, all proteins were passed through a Superdex S-75 column (GE Healthcare) with buffer A. The molecular mass of all four proteins was estimated in buffer A using a Superdex S-75 column and calibrated with gel filtration standard markers [Bio-Rad: γ-globulin (bovine), 158 kDa; ovalbumin (chicken), 44 kDa; myoglobin (horse), 17 kDa; and vitamin B12, 1.35 kDa]. The molecular mass of NLP7 PB1 was also determined using a Superdex S-75 column and calibrated with gel filtration standard markers in buffer C [20 mM Tris, pH 8.8, 0.5 M NaCl, 10% glycerol, 5 mM reduced glutathione (GSH), 1 mM tris(2-carboxyethyl)phosphine (TCEP)]. Buffer C was designed to reduce protein aggregation and increase the solubility of the protein samples. After SEC, all proteins were concentrated using Amicon Ultra-0.5 centrifugal filters (Merck, Darmstadt, Germany) and quantified using a DS-11 spectrophotometer (DeNovix).
Superdex s75 column
Manufactured by GE Healthcare
Sourced in United States
The Superdex S75 column is a size exclusion chromatography column designed for the separation and purification of proteins, peptides, and other biomolecules. It features a dextran-based matrix that allows for efficient separation based on molecular size and shape.
Automatically generated - may contain errors
Lab products found in correlation
Nickel affinity chromatography, by Qiagen (2 mentions)
Bl21 de3 ril, by Agilent Technologies (2 mentions)
8 ml nickel column, by Qiagen (2 mentions)
Petduet vector, by Merck Group (2 mentions)
Optilab t rex interferometric refractometer, by Wyatt Technology (2 mentions)
Astra 6, by Wyatt Technology (2 mentions)
Dawn heleos 2, by Wyatt Technology (2 mentions)
Instantblue, by Abcam (2 mentions)
Amicon ultra centrifugal filter unit, by Merck Group (1 mentions)
Ni nta agarose bead, by Qiagen (1 mentions)
Histrap nickel column, by GE Healthcare (1 mentions)
Myoglobin, by GE Healthcare (1 mentions)
Ovalbumin (ova), by GE Healthcare (1 mentions)
Amicon ultra 0.5 centrifugal filter, by Merck Group (1 mentions)
Ds 11 spectrophotometer, by DeNovix (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Superdex 75 increase 10 300 gl, by GE Healthcare (1 mentions)
Quick start bradford assay, by Bio-Rad (1 mentions)
Ni2 nta resin, by GE Healthcare (1 mentions)
Glutathione (gsh), by GE Healthcare (1 mentions)
23 protocols using superdex s75 column
1
Protein Purification using Size-Exclusion Chromatography
2
Recombinant Expression and Purification of Zebrafish TRAF6 TRAF-C Domain
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Zebrafish TRAF6 TRAF-C domain (370–525) gene sequence was synthesized (IDT) and cloned into a modified pET16 vector with an N-terminal 6 × His-MBP-SUMO2-tag. Expression was performed in E. coli BL21-RIL DE3 (Agilent) bacteria harboring a pRARE2 tRNA plasmid using MDG media in pre-cultures and M9ZB media in large-scale cultures (2 × 1 L). Cells were induced with 0.5 mM IPTG after reaching an OD600 1.0–1.5 and protein was expressed at 18 °C for 18 h. Harvested cells were re-suspended in lysis buffer (20 mM HEPES-KOH pH 7.5, 400 mM NaCl, 30 mM imidazole, 10% glycerol, 1 mM DTT) and disrupted by sonication. Lysate was clarified by centrifugation and subsequent filtration through glass wool. Recombinant TRAF6 TRAF-C domain protein was purified by nickel-affinity chromatography (QIAGEN) and the His-MBP-SUMO2-tag was subsequently removed by addition of human SENP2-protease (250 mg, fragment D364-L589 with M497A mutation) and dialyzed over night at 4 °C in 20 mM HEPES-NaOH pH 7.5, 200mMNaCl, 1mMDTT. The protein was further purified by size exclusion chromatography using a Superdex S75 column (GE Healthcare) equilibrated with 20mMHEPES-NaOH pH 7.5, 150mMNaCl, 1mMTCEP. Final TRAF6 TRAF-C domain protein was concentrated to 5–6 mg mL−1 before being flash-frozen in liquid nitrogen for storage at −80 °C.
3
Purification of HNF4α LDB Domain
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The LDB domain of HNF4α (aa. 148–377) subcloned into pNIC28-Bsa4 was expressed in E. coli Rosetta. The recombinant protein harboring an N-terminal His6 tag was initially purified by Ni2+-affinity chromatography. Size exclusion chromatography was performed using Superdex S75 column (GE Healthcare, Chicago, IL, USA.), and the pure protein was stored in a buffer containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5, 200 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), and 5% w/v glycerol.
4
Producing Holo-Acyl Carrier Protein
5
Expression and Purification of AmSOD
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Chemically competent E. coli BL21(DE3) Rosetta cells (Bio-Bharati LifeScience Pvt. Ltd., Kolkata, India) was transformed with Am_SOD ORF- pET22b+ construct and selected on LB agar plate containing 100 µg ml−1 of ampicillin and 34 µg ml-1 of chloramphenicol. Am_SOD expression was induced using 0.5 mM of IPTG at 16 °C for 12 h and purified under native condition using Ni-NTA agarose beads (QIAGEN) packed in polypropylene-made gravity column following manufacturer’s protocol. Ni-NTA purified protein was refractionated in Superdex S75 column (GE Lifesciences). Fractions containing Am_SOD with >95% purity were concentrated in Amicon® Ultra Centrifugal Filter Units, 10 kDa cut-off (Merck-Millipore). Am_SOD (1 µg) was run in reducing and non-reducing SDS- PAGE.
6
Recombinant Expression and Purification of Zebrafish TRAF6 TRAF-C Domain
7
Expression and Purification of Holo-ACP
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Expression and purification of holo-ACP has been described previously [11 (link)]. Briefly, the acyl-carrier-protein and holo-(acyl-carrier-protein) synthase genes were cloned into a pETDUET vector (EMD-Millipore). Both proteins were expressed simultaneously following the same steps as FabD expression (above). After lysing and centrifuging the cells the clarified lysate was applied to an 8 ml nickel column (Qiagen) and an imidazole gradient was run from 0–500 mM imidazole in buffers containing 20 mM Tris-HCl, 500 mM sodium chloride, pH 8.1. Fractions containing holo-ACP were diluted to reduce sodium chloride to 100 mM and loaded onto a 110 ml Q-Sepharose column. A linear gradient from 200–600 mM potassium chloride was applied to the column and the holo-ACP eluted around 500 mM potassium chloride. Fractions containing ACP were concentrated and exchanged into a buffer containing 25 mM MOPS, 100 mM sodium chloride, 1 mM DTT, pH 7.1 using a Superdex S75 column (GE). Fractions containing holo-ACP were concentrated using a 3 kDa amicon filter unit and stored at −80°C in 10% glycerol. The ACP extinction coefficient used for quantification was 1490 M-1cm-1.
8
Nanobody Production and Characterization
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The purified human TorsinAEQ-LULL1 complex was injected into a male alpaca (Lama pacos) for immunization. Generation and screening of nanobodies was carried out as previously described (Sosa et al., 2014 (link)). Each of the selected nanobodies was subcloned into a pET-30b(+) vector with a C-terminal His6-tag. Each nanobody was bacterially expressed and Ni2+-affinity purified essentially as described (see above). Different from the TorsinA-containing preparations, MgCl2 and ATP were eliminated from all buffer solutions. The Ni2+-eluate was purified via size exclusion chromatography on a Superdex S75 column (GE Healthcare) in running buffer (10 mM HEPES/NaOH pH 8.0, 150 mM NaCl). Nanobody binding was validated by size exclusion chromatography on a 10/300 Superdex S200 column in 10 mM HEPES/NaOH pH 8.0, 150 mM NaCl, 10 mM MgCl2 and 0.5 mM ATP. Equimolar amounts of TorsinAEQ-LULL1 and TorsinAEQ-LULL1-VHH were loaded and nanobody binding was monitored by a shift in the elution profile and via SDS-PAGE analysis. After validating VHH-BS2 interaction with TorsinAEQ-LULL1, the C-terminal His6-tag of VHH-BS2 was removed from the pET-30b(+) vector for co-purification experiments.
9
Purification of Bromodomain Proteins
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Purified proteins VBC, SMACRC2BD (bromodomain), BRD4BD1 or BRD4BD2 were expressed and purified according to the previously published protocols28 (link),31 (link). Briefly, purification of N-terminal His6 tagged proteins (1) SMARCA2BD 1373–1511 ∆1400–1417; (2) VHL (54-213), ElonginB (17-112) ElonginC (1-104); and (3) BRD4 (44-168) cells were lysed by 2 passes on microfluidizer and ultracentrifuged at 235,000 g for lysate clarification. Clarified lysate was mixed with Talon resin and nutated for an hour at 4 °C, washed and eluted with 250 mM imidazole. The His6 tag was removed using TEV protease. Cleaved proteins were then purified using Superdex S-75 column (GE Healthcare). Purified protein samples were stored in 10 mM HEPES 7.5, 150 mM NaCl, 0.5 mM TCEP at -80 °C to be used for crystallization/SPR studies.
10
Expression and Purification of CALP Proteins
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CALP and CALPC319A [25 ] and CALP-His10 [20 (link)] proteins were expressed and purified as previously described. Cer-CALP vector was transformed into BL21(DE3) RIL cells and protein was expressed essentially as described for Na+/H+ Exchanger Regulatory Factor (NHERF)1 PDZ1 [21 (link)]. Briefly, cells were induced for ~16 h at 20°C and harvested by centrifugation. Following lysis by French press, the lysate was clarified by ultracentrifugation and purified using a HisTrap nickel column (GE Healthcare), and then further purified using size-exclusion chromatography (SEC) with a Superdex S75 column (GE Healthcare) equilibrated in 25 mM Tris (pH 8.5), 150 mM NaCl, 0.1 mM tris(2-carboxyethyl)phosphine (TCEP), 0.1 mM ATP, 0.02% (w/v) NaN3. The protein was dialyzed into 25 mM sodium phosphate (pH 7.4), 150 mM NaCl, 0.1 mM TCEP, and 5% (v/v) glycerol for cryostorage.
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