HEK293T cells stably expressing His-tagged Ub/Ub-ΔGG or SUMO/SUMO-ΔGG were treated with or without 4 mM HU for 3 h. For the ubiquitination assay, cells were pre-treated with 10 μM MG132 for 1 h before HU treatment. Cells were then collected, lysed, and sonicated in Buffer A (6 M guanidine–HCl, 100 mM NaH2PO4/Na2HPO4 and 15 mM imidazole). Clarified lysates were incubated with cobalt resin (Thermo Scientific) at 4°C overnight. The beads were then centrifuged and washed once with Buffer A, once with Buffer B (25 mM Tris–HCl [pH 6.8] and 10 mM imidazole). After washing, the resin-bound proteins were eluted by boiling in 2× SDS loading buffer supplemented with 250 mM imidazole and analyzed by SDS-PAGE. For the SUMOylation assay, cells were harvested and lysed in Buffer I (6 M guanidine–HCl, 100 mM NaH2PO4/Na2HPO4, 10 mM Tris–HCl [pH 8.0], and 5 mM imidazole). After sonication, the lysates were incubated with cobalt resin (Thermo Scientific) at 4°C overnight. Bound complexes were washed once each in Buffer I, Buffer II (8 M urea, 100 mM NaH2PO4/Na2HPO4 and 10 mM Tris–HCl [pH 8.0]), Buffer III (8 M urea, 100 mM NaH2PO4/Na2HPO4 and 10 mM Tris–HCl [pH 6.3]), and Buffer IV (25 mM Tris–HCl [pH 6.8] and 10 mM imidazole). The protein-bound beads were boiled in 2× SDS loading buffer supplemented with 250 mM imidazole and then subjected to SDS-PAGE.
Cobalt resin
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Cobalt resin is a type of chromatography resin designed for the purification of proteins and other biomolecules. It operates based on the principle of metal affinity chromatography, utilizing the interaction between cobalt ions and specific amino acid residues in proteins to facilitate their separation and isolation.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Superdex 75, by GE Healthcare (1 mentions)
Coomassie brilliant blue, by Bio-Rad (1 mentions)
Mwco slide a lyzer dialysis cassette, by Thermo Fisher Scientific (1 mentions)
E coli bl21 strains, by RBC Bioscience (1 mentions)
Ni nta resin, by Qiagen (1 mentions)
Expi293 cell, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose bead, by GE Healthcare (1 mentions)
Streptavidin conjugated beads, by GE Healthcare (1 mentions)
Bl21 de3 e coli strain, by New England Biolabs (1 mentions)
Glutathione sepharose 4 fast flow medium, by GE Healthcare (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
11 protocols using cobalt resin
1
Purification of Ubiquitinated and SUMOylated Proteins
2
Activation of Recombinant Gingipain Protease
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Initially, VAMP8-modified 293 cells were washed with PBS and lysed with IGEPAL CA-630 buffer containing protease and phosphatase inhibitors. His-tagged Lys-gingipain recombinant protein (enQuireBioReagents, Littleton, CO, USA) was incubated with cobalt resin (Thermo Fisher Scientific) and IGEPAL CA-630 buffer at 4 °C for 2 h. The supernatant was removed and gingipain activation buffer54 (200 mmol·L-1 HEPES [pH 8.0], 5 mmol·L-1 CaCl2, and 20 mmol·L-1 L-cysteine, HCl solution) were added and incubated at 37 °C for 30 min. The samples were washed thrice with IGEPAL CA-630 buffer and 1× SDS sample buffer and processed for immunoblotting.
3
Purification of Recombinant His-Tagged Proteins
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The N terminus 6 × His expression constructs were transformed into Escherichia coli strain BL21. Protein expression was induced with 1 mM IPTG at OD600 of 0.6-0.8 and the culture was incubated at 18 °C overnight. Soluble proteins were loaded in a column chromatography cartridge packed with cobalt resin (ThermoScientific), washed with a washing buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole; pH 7.4), and eluted with an elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 150 mM imidazole; pH 7.4). Affinity-purified recombinant proteins were dialyzed against a PBS (pH 7.2) buffer at 4 °C overnight and the dialyzed proteins were concentrated using either 10 K or 30 K centrifugal filter units (Merck). Concentrated BcATG4 and BcATG8 were gel filtrated using a Superdex 200 and Superdex 75 (GE healthcare) on an AKTA explorer system, respectively. Purified proteins were quantified using BSA (ThermoScientific) as a standard and confirmed by SDS-PAGE after Coomassie brilliant blue staining (Bio-rad).
4
Recombinant Protein Purification from E. coli
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BL21 E. coli strains (RBC Bioscience, Taipei, Taiwan) were transformed with the pET-DEST42-Rho-OR2AT4-6xHis plasmid and incubated in Luria-Bertani (LB) broth (BD Difco, Sparks, MD, USA) containing 100 μg/mL ampicillin. The cells were grown at 37 °C with shaking at 200 rpm until the optical density at 600 nm reached to 0.6–0.8. Protein expression was induced with 1 mM IPTG at 16 °C for 16 h at 200 rpm. Cells were harvested at 6000×g for 10 min and lysed by sonication for 20 min (5 s on/off). Homogenized cells were centrifuged (12,000×g, 30 min) and separated into soluble/insoluble fractions. Insoluble fraction was solubilized in solubilization buffer (0.1 M Tris-HCl, 20 mM SDS, 1 mM EDTA, and 100 mM DTT) and dialysis by 10 kDa MWCO Slide-A-Lyzer Dialysis Cassette (Thermo Fisher Scientific, Waltham, MA, USA) in 0.1 M sodium phosphate buffer (pH 8.0) containing 10 mM SDS for 4 h. Solubilized proteins were incubated with cobalt resin (Thermo Fisher Scientific) or Ni-NTA resin (Qiagen, Crawley, UK) for 4 h at room temperature for shaking. His-tagged protein was passed through filter column (Bio-rad, Richmond, CA, USA) and eluted from the 0.1 M sodium phosphate buffer (pH 6.0) with 10 mM SDS by gravity flow. Eluted protein was performed to western blotting and Coomassie blue staining.
5
Expi293 Protein Expression and Purification
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Expi293 cells (Life Tech) were passaged in Expi293T serum-free medium (Life Tech) and used for protein expression. The expression plasmids were transfected into Expi293 cells via lipid transfection (Fectopro, Polyplus), and the cell culture medium containing secreted protein was collected 4 days later. Following sterile filtration, 6xHis tagged proteins were purified via immobilized metal-affinity chromatography (IMAC). Briefly, proteins were batch adsorbed to cobalt resin (Thermo Scientific) overnight at 4C, washed with 10 column volumes of phosphate buffered saline, then eluted with 500mM imidazole. Proteins were dialyzed into HEPES buffered saline overnight, concentrated (Centricon), and frozen at -80C until use.
6
Purification and Analysis of Ubiquitylated Proteins
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When indicated, ubiquitylated proteins were purified by histidine (His) pull-down. To this end, cells expressing His-tagged ubiquitin were lysed in denaturing buffer, containing 8 m urea, 100 mm Na2PO4, 10 mm Tris-HCl, pH 8.0. Lysates were applied to cobalt resin (Thermo Fisher Scientific) for 2 h, and then beads were washed twice with buffer containing 8 m urea, 100 mm Na2PO4, 10 mm Tris-HCl, pH 6.3; denatured in NuPAGE sample buffer; and loaded on a 6% polyacrylamide gel.
GST and GST-Pdcd4 recombinant proteins were produced in Escherichia coli BL21, as described previously (34 (link)). For GST pull-down, HEK293T cells (3 × 106) were transfected with pCMV6-IBtkα-FLAG or IBtkα-FLAG mutant plasmids (4 μg), and 48 h later, cells were lysed in RIPA buffer. The lysate (1 mg) was incubated for 1 h with GST or GST-Pdcd4-WT and mutants at 4 °C under constant agitation. Then glutathione-Sepharose beads (30 μl) (GE Healthcare) were added to the mix for a 1-h incubation at 4 °C. Subsequently, beads were washed three times with RIPA buffer, and the bound proteins were eluted from beads by boiling in SDS sample buffer. Protein samples were subjected to electrophoresis on NuPAGE 4–12% polyacrylamide gel and analyzed by immunoblotting with the appropriate antibodies.
GST and GST-Pdcd4 recombinant proteins were produced in Escherichia coli BL21, as described previously (34 (link)). For GST pull-down, HEK293T cells (3 × 106) were transfected with pCMV6-IBtkα-FLAG or IBtkα-FLAG mutant plasmids (4 μg), and 48 h later, cells were lysed in RIPA buffer. The lysate (1 mg) was incubated for 1 h with GST or GST-Pdcd4-WT and mutants at 4 °C under constant agitation. Then glutathione-Sepharose beads (30 μl) (GE Healthcare) were added to the mix for a 1-h incubation at 4 °C. Subsequently, beads were washed three times with RIPA buffer, and the bound proteins were eluted from beads by boiling in SDS sample buffer. Protein samples were subjected to electrophoresis on NuPAGE 4–12% polyacrylamide gel and analyzed by immunoblotting with the appropriate antibodies.
7
Enrichment of Ubiquitinated PCNA
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HEK293T cells stably expressing His-tagged wild-type Ub or Ub-ΔGG were transfected with plasmids encoding SFB-tagged PCNA. After 24 h of transfection, cells were treated with 2 mM HU and 2 mM ATRi for 2 h. Subsequently, cells were collected, lysed, and sonicated in denaturing Buffer A (6 M guanidine–HCl, 100 mM NaH2PO4/Na2HPO4, and 15 mM imidazole). Clarified lysates were incubated with cobalt resin (Thermo Scientific) at 4 °C overnight. The beads were then centrifuged and washed once with Buffer A, once with Buffer B (25 mM Tris-HCl [pH 6.8] and 10 mM imidazole), and the bound ubiquitinated proteins were eluted using NETN buffer containing 200 mM imidazole for 1 h. The eluent was incubated with streptavidin-conjugated beads (GE Healthcare) for 1 h at 4 °C. The beads were washed with NETN buffer containing 300 mM NaCl three times, and then the ubiquitinated SFB-tagged PCNA was eluent with NETN buffer containing 1 mg/mL biotin (Sigma-Aldrich).
8
Recombinant VHH Production in E. coli
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Chromatibody or control VHH were produced in the BL21 DE3 E. coli strain (New England Biolabs). 2xTY medium, supplemented with 100 µg/ml ampicillin and 0.1% glucose, was inoculated with a fresh colony, and the culture was grown at 37°C under agitation (220 rpm). When the optical density at 600 nm (OD600) reached 0.7, induction was performed (0.5 mM IPTG) at 28°C for 16 h. The VHHs were purified from periplasmic extract or directly from the culture supernatant in a two-step process involving an ammonium sulfate precipitation (65% saturation) followed by an immobilized metal affinity chromatography (cobalt resin, Thermo Scientific). After imidazole elution, the buffer was exchanged to PBS by dialysis, and the concentration of the purified VHH determined by Nanodrop (Thermo Scientific).
9
Expression and Purification of Nanobodies
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Nb DNA constructs were transformed into BL21(DE3) cells and plated on Agar with 50 μg/mL ampicillin at 37°C overnight. Cells were cultured in an LB broth to reach an O.D. of ∼0.5–0.6 before IPTG (0.5–1 mM) induction at 16/20°C overnight. Cells were then harvested, sonicated, and lysed on ice with a lysis buffer (1xPBS, 150 mM NaCl, 0.2% TX-100 with protease inhibitor). After cell lysis, protein extracts were collected by centrifugation at 21,000 x g for 10 mins and the his-tagged Nbs were purified by the Cobalt resin (Thermo) and natively eluted with a buffer containing 150 mM imidazole buffer. Eluted Nbs were subsequently dialyzed in a dialysis buffer (e.g., 1x DPBS, pH 7.4 or SEC buffer).
For the periplasmic preparation of Nbs (2–31 and 2–45), cell pellets were resuspended in the TES buffer (0.1 M Tris-HCl, pH 8.0; 0.25 mM EDTA, pH 8.0; 0.25 M Sucrose) and incubated on ice for 30 min. The supernatants were collected by centrifugation and subsequently dialyzed to DPBS. The resulting Nbs were then purified by Cobalt resin as described above.
For the periplasmic preparation of Nbs (2–31 and 2–45), cell pellets were resuspended in the TES buffer (0.1 M Tris-HCl, pH 8.0; 0.25 mM EDTA, pH 8.0; 0.25 M Sucrose) and incubated on ice for 30 min. The supernatants were collected by centrifugation and subsequently dialyzed to DPBS. The resulting Nbs were then purified by Cobalt resin as described above.
10
Recombinant Protein Expression and Purification
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Recombinant proteins were expressed using the E. coli (BL21, Arctic strain) system after induction by 0.25 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 13°C overnight as described elsewhere.34 (link),37 (link),38 (link) Soluble Hisx6-tagged proteins were purified using the Cobalt resins (Thermo Fisher Scientific), while GST and GST-tagged protein were isolated using the Glutathione Sepharose 4 Fast Flow medium (GE Healthcare Life Sciences) according to the instructions of the manufacturers. The tag-free S-αTSR protein was purified by a two-step procedure, including a precipitation of the target protein from the bacteria lysate using ammonium sulfate [(NH4)2SO4], and an ion exchange chromatography (see below).35 (NH4)2SO4 at 1.6 M end concentration was used to effectively precipitate the tag-free S-αTSR protein at room temperature (20–22°C).
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