Recombinant ANXAs were produced and purified as previously described (7 (link), 8 (link)). Briefly, ANXA cDNA constructs were subcloned into the bacterial expression vector pETM11-SUMO3 (originally from EMBL Protein Expression and Purification Core Facility) with or without C-terminally tagged superfold GFP (sfGFP) and N-terminally tagged with a His6-tag and a SUMO3 domain. BL21 (DE3) competent Escherichia coli cells were used as expression hosts for the production of recombinant proteins, and the expressions were induced overnight at 18 °C using Isopropyl β-D-1 thiogalactopyranoside (IPTG). Cells were harvested by centrifugation and lysed mechanically by sonication. His6-tagged proteins were purified using Immobilized Metal-Affinity Chromatography (IMAC) with Ni-NTA (nickel-nitrilotriacetic acid) resins (Qiagen). The His6-tag and SUMO3 domain were cleaved of by SUMO-Specific Protease 2 (SENP2) (200:1) followed by dialysis overnight at 4 °C. Proteins were further purified and separated using Fast Protein Liquid Chromatography (FPLC) on a Superdex 200 size-exclusion chromatography column (SuperdexTM 200, 10/300 GL, GE Healthcare Life Sciences). Protein fractions were collected and stored at −80 °C until use.
Superdex 200
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Superdex 200 is a size-exclusion chromatography medium used for the separation and purification of proteins, peptides, and other biomolecules. It is composed of highly cross-linked agarose beads that allow for efficient separation based on molecular size. The Superdex 200 matrix provides a wide fractionation range and high resolution, making it a versatile tool for a variety of applications in biotechnology and life science research.
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Lab products found in correlation
Superdex 75, by GE Healthcare (35 mentions)
Ni nta resin, by Qiagen (14 mentions)
Mono q, by GE Healthcare (12 mentions)
Hitrap q hp, by GE Healthcare (12 mentions)
Ni nta, by Qiagen (11 mentions)
Hitrap mabselect sure, by GE Healthcare (11 mentions)
Biomax sk membrane, by Merck Group (10 mentions)
Ni nta agarose, by Qiagen (8 mentions)
Resource q, by GE Healthcare (8 mentions)
Nanodrop, by Thermo Fisher Scientific (8 mentions)
Co nta agarose, by Takara Bio (7 mentions)
Gel filtration standard, by Bio-Rad (7 mentions)
Q sepharose fast flow, by GE Healthcare (7 mentions)
Ni nta column, by Qiagen (6 mentions)
Amylose resin, by New England Biolabs (6 mentions)
Superose 6, by GE Healthcare (6 mentions)
Bac to bac system, by Thermo Fisher Scientific (6 mentions)
Amicon ultra, by Merck Group (6 mentions)
Ni nta column, by Takara Bio (5 mentions)
Complete edta free, by Roche (5 mentions)
746 protocols using superdex 200
1
Recombinant Annexin Protein Production
2
Keap1 Protein Purification and Crystallization
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The
DNA coding domain for human Keap1 (6 ×
His- TCS-Keap1 (A321-T609) [E540A, E542S]) was cloned into pET24 and
expressed in Escherichia coli BL21(DE3)-Star
in LB media at 291 K. After harvest, the cells were resuspended in
20 mM Tris/HCl, pH 7.5, 200 mM NaCl, 5% glycerol, 1 mM DTT, protease
inhibitors, disrupted with a high-pressure homogenizer, and clarified
by centrifugation. Purification was made by affinity chromatography
using Ni Sepharose FF (GE Healthcare), eluted in one step with 300
mM imidazole followed by SEC (Superdex200, GE Healthcare). The purified
protein was detagged at room temperature with a thrombin cleave kit
(Sigma) and finalized by a second SEC step (Superdex200, GE Healthcare)
in buffer 20 mM Tris/HCl, pH 7.5, 5 mM DTT before concentration (Amicon
cells, 10 kDa cutoff). Crystals were grown at 20 °C in sitting
drops using 200 nL of protein (11–13 mg/mL) and 200 nL of well
solution (3.7–4.1 M ammonium acetate, 0.09 M sodium acetate
pH 4.6, and 10 mM cadmium chloride). Crystals appeared after one day
but continued to grow for approximately one week. Compound14 was soaked by incubating crystals for 1 h with 1 mM compound in
5 M ammonium acetate and 0.1 M sodium acetate at pH 4.6. Crystals
were subsequently frozen in liquid nitrogen using a soaking solution
supplemented with 20% glycerol as the cryoprotectant prior to data
collection.
DNA coding domain for human Keap1 (6 ×
His- TCS-Keap1 (A321-T609) [E540A, E542S]) was cloned into pET24 and
expressed in Escherichia coli BL21(DE3)-Star
in LB media at 291 K. After harvest, the cells were resuspended in
20 mM Tris/HCl, pH 7.5, 200 mM NaCl, 5% glycerol, 1 mM DTT, protease
inhibitors, disrupted with a high-pressure homogenizer, and clarified
by centrifugation. Purification was made by affinity chromatography
using Ni Sepharose FF (GE Healthcare), eluted in one step with 300
mM imidazole followed by SEC (Superdex200, GE Healthcare). The purified
protein was detagged at room temperature with a thrombin cleave kit
(Sigma) and finalized by a second SEC step (Superdex200, GE Healthcare)
in buffer 20 mM Tris/HCl, pH 7.5, 5 mM DTT before concentration (Amicon
cells, 10 kDa cutoff). Crystals were grown at 20 °C in sitting
drops using 200 nL of protein (11–13 mg/mL) and 200 nL of well
solution (3.7–4.1 M ammonium acetate, 0.09 M sodium acetate
pH 4.6, and 10 mM cadmium chloride). Crystals appeared after one day
but continued to grow for approximately one week. Compound
5 M ammonium acetate and 0.1 M sodium acetate at pH 4.6. Crystals
were subsequently frozen in liquid nitrogen using a soaking solution
supplemented with 20% glycerol as the cryoprotectant prior to data
collection.
3
Purification of Nuclear Transport Proteins
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GST-NP ncNLS, GST-SV40 NLS40 (link), and His/S-ΔIBB importin-α1 (mouse, residues 70–529)19 (link) were expressed from pGEX-TEV, pGEX-4T-1, and pET30a, respectively, in E. coli strain BL21-CodonPlus(DE3)RIL (Stratagene). GST-NLS fusion proteins were purified over Glutathione-sepharose 4B (GE Healthcare) and gel filtration over Superdex200 (GE Healthcare). His/S-ΔIBB importin-α1 was purified over Ni-NTA (Novagen) and gel filtration over Superdex200 (GE Healthcare). Mutants were created using the Quickchange system (Stratagene), and all constructs were verified by DNA sequencing.
4
Purification of p97, Ufd1, and NPL4 Proteins
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All proteins were expressed in E. coli BL21 (DE3) cells (Novagen). p97-His (pET28a vector) and Ufd1-His (pET28a vector) expression were induced by 1 mM IPTG (Life Technologies) at an OD600 of 0.6 for 10 hours at 22°C. NPL4 WT and MUT (pGEX-2TK) were induced by 0.4 mM IPTG at an OD600 of 0.8 overnight at 16°C. In case of p97 and UFD1, bacterial pellet was suspended in buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 2.5 mM MgCl2, 20 mM imidazole, 5% glycerol) and lysed by sonication and centrifuged (14000G for 20 minutes). Proteins were purified by Ni-NTA chromatography (Qiagen) according to manufacturer instructions. In case of p97, the protein was further purified by gel filtration (Superdex 200, GE Healthcare). In case of WT and MUT GST-NPL4, bacterial pellet was suspended in phosphate buffer (PBS, 0.1% Triton-X100, 300 mM NaCl) and lysed by sonication and centrifuged (14000G for 10 min). Proteins were purified by glutathione sepharose 4B (Life Technologies) according manufacturer’s protocol. The proteins were further purified by gel filtration (Superdex 200, GE Healthcare).
5
Size Exclusion Chromatography of tPA
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Size exclusion chromatography was performed using a Superdex 200 column (GE Healthcare) as described previously [33 (link)]. The protein of interest or molecular mass standards were applied to the Superdex 200 (GE Healthcare) column, and eluted using 50 mM Tris–HCl pH 8.0 and 150 mM NaCl. The protein molecular weight standards used were albumin bovine V (66.2 kDa), chicken egg albumin (44.2 kDa), chymotrypsinogen A (24.5 kDa), and lysozyme (14.4 kDa). The peak elution volumes (measured at 280 nm) were used to calculate the standard curve using the equation log MW = −0.01712 Ve + 6.05667, with R2 = 0.9726. tPA was diluted to 1 mg ml−1 in buffer (50 mM Tris–HCl pH 8.0 and 150 mM NaCl), and then loaded on the column. tPA was eluted as a single peak in a volume of 72.1 ml, corresponding to an estimated molecular weight of 66.68 kDa.
6
Purification of VDAC-Containing Nanodiscs
7
Purification of p97, Ufd1, and NPL4 Proteins
8
Histone Octamer Dissociation Dynamics
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Histone octamer was assembled from individual histone proteins as previously described with modification [7] (link). Prior to Superdex 200 chromatography (16 × 600 mm; GE Healthcare), H2A-N38C histone octamer was crosslinked by dialysis into crosslinking buffer [20 mM Tris–HCl (pH 9.0), 2 M NaCl, 1 mM EDTA, 1 mM oxidized glutathione] for 24 h at 22 °C in the dark. This step was omitted for the H3-R40C and H2A-N38C/H3-R40C histone octamers, which were instead kept reduced with 1 mM DTT during size-exclusion chromatography. All octamers included the histone H3-C110A substitution so as not to interfere with disulfide formation.
For histone octamer dissociation studies, samples already purified by Superdex 200 chromatography were dialyzed into either 50 mM sodium acetate (pH 5.0) or 10 mM Tris–HCl (pH 7.5), and 0.4 M, 0.8 M or 2 M NaCl and 1 mM EDTA for 12 h. Each sample (1 mg) was injected onto a Superdex 200 column (10 × 300 mm; GE Healthcare). Fractions were analyzed by SDS PAGE (18% acrylamide). Histone band intensities were quantified by densitometry (Gel Logic, Carestream). Protein markers were Precision Plus Protein Standard (Bio-Rad).
For histone octamer dissociation studies, samples already purified by Superdex 200 chromatography were dialyzed into either 50 mM sodium acetate (pH 5.0) or 10 mM Tris–HCl (pH 7.5), and 0.4 M, 0.8 M or 2 M NaCl and 1 mM EDTA for 12 h. Each sample (1 mg) was injected onto a Superdex 200 column (10 × 300 mm; GE Healthcare). Fractions were analyzed by SDS PAGE (18% acrylamide). Histone band intensities were quantified by densitometry (Gel Logic, Carestream). Protein markers were Precision Plus Protein Standard (Bio-Rad).
9
Purification of Recombinant Proteins
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All proteins were expressed in BL21-CodonPlus (DE3)-RIPL (Agilent) or BL21 E. coli strains (NEB). CytATL, cytATL(R232Q), and MBP-cyclin B∆90 were purified, as described previously (Wang et al., 2013 (link)). CytATL-GFP, cytATL(R232Q)-GFP and all cytLnp proteins except cytLnp-SBP were isolated using a Ni-NTA resin (Thermo Scientific), followed by anion-exchange chromatograph (HiTrap Q HP, GE Healthcare) and gel filtration (Superdex 200, GE Healthcare). CytLnp-SBP was purified using a Ni-NTA resin, followed by purification on a streptavidin resin (Thermo Scientific) and gel filtration (Superdex 200, GE Healthcare). The proteins were snap-frozen in 20 mM HEPES pH 7.5, 150 mM KCl, 250 mM sucrose, and 1 mM dithiothreitol (DTT). For the gel filtration analysis in Figure 11—figure supplement 1F , 400 µg of purified His6-cytLnp/-N1/-N2/-C in 200 µL were injected onto a Superdex 200 column in buffer containing 20 mM HEPES pH 7.5, 150 mM KCl, and 1 mM DTT.
10
Purification of TTC7B/FAM126A Complex
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