ADAT2/ADAT3 WT and mutants were produced by co-expression in Escherichia coli BL21(DE3) cells in LB Broth medium. Culture induction was performed at 22°C by adding final concentration of 0.5 mM of isopropyl-1-thio-β-D-galactopyranoside (IPTG) in presence of 100 μM of Zn(SO4)2. Cells were harvested, resuspended and lysed in a buffer containing 10 mM Tris-HCl pH 8.0 and 200 mM NaCl and centrifuged at 17 500 rpm for 1 h at 4°C. The supernatant was incubated with Talon Affinity resin (Clonetech). To release the his-tagged complex from the Talon resin, the sample was treated with 3C protease overnight at 4°C. The next day, ion exchange chromatography was performed with a HiTrap Q HP column (GE Healthcare) using a gradient of NaCl from 50 mM to 1 M NaCl to remove bound nucleic acids. The sample was then further purified by size exclusion chromatography in 10 mM Tris HCl pH 8.0, 200 mM NaCl and 0.5 mM TCEP on a 16/60 Superdex 200 gel filtration column (GE Healthcare). Escherichia coli TadA was produced and purified using the same protocol as for the ADAT complex.
Talon affinity resin
Manufactured by Takara Bio
Sourced in United States
TALON affinity resin is a cobalt-based metal affinity resin used for the purification of histidine-tagged recombinant proteins. The resin binds to the histidine tag present on the target protein, allowing for its separation and purification from other cellular components.
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Lab products found in correlation
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (2 mentions)
Hitrap q hp column, by GE Healthcare (1 mentions)
Nhs biotin, by Thermo Fisher Scientific (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Pdonr221, by Thermo Fisher Scientific (1 mentions)
Gateway lr reaction, by Thermo Fisher Scientific (1 mentions)
Superdex s200 gel filtration column, by GE Healthcare (1 mentions)
Arcticexpress de3 rp, by Agilent Technologies (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose, by GE Healthcare (1 mentions)
Hrp conjugated goat anti mouse igg antibody, by Santa Cruz Biotechnology (1 mentions)
Versadoc gel imaging system, by Bio-Rad (1 mentions)
Mouse anti rev antibody, by Santa Cruz Biotechnology (1 mentions)
Bl21 codonplus de3 ril cells, by Agilent Technologies (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Nanodrop, by DeNovix (1 mentions)
Imidazole, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
E coli bl21 λ de3, by Agilent Technologies (1 mentions)
19 protocols using talon affinity resin
1
Purification of ADAT2/ADAT3 Complex and TadA from E. coli
2
Purification of Apical Protein Domains
3
Nanobody Expression, Purification, and Biotinylation
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Selected nanobody constructs were expressed and purified from SHuffle T7 E. coli (New England Biolabs). An overnight culture was diluted 1:100 into fresh Luria Broth with appropriate antibiotics and grown at 37°C until a OD600 of ~1.2 was reached. The temperature was lowered to 18°C, 1 mM IPTG was added, and protein expression was continued overnight. Cells were harvested by centrifugation and lysed with an Emulsiflex. Nanobodies were purified from clarified supernatants using Talon affinity resin (Takara). Eluted nanobodies were concentrated in an Amicon centrifugal spin concentrator (3K MWCO), then run over an S-75 size exclusion column equilibrated in PBS pH 7.4. Fractions were analyzed by SDS-PAGE, pooled, snap-frozen in liquid N2, and stored at -70 °C until use.
Selected nanobodies with C-terminal Avi-tags were biotinylated as described [44 (link)]. Briefly, a sample of Avi-tagged nanobody (100 μM) was incubated with 10 mM MgCl2, 10 mM ATP, 1/10th mass BirA (Addgene plasmid 20857), and 50 μM D-biotin at 30 °C for 1 hr. Samples were further purified over an S-75 size exclusion column equilibrated in PBS pH 7.4. Fractions containing biotinylated nanobody were analyzed by SDS-PAGE, and nanobody-containing fractions (>90% purity) were stored at -20 °C.
Selected nanobodies with C-terminal Avi-tags were biotinylated as described [44 (link)]. Briefly, a sample of Avi-tagged nanobody (100 μM) was incubated with 10 mM MgCl2, 10 mM ATP, 1/10th mass BirA (Addgene plasmid 20857), and 50 μM D-biotin at 30 °C for 1 hr. Samples were further purified over an S-75 size exclusion column equilibrated in PBS pH 7.4. Fractions containing biotinylated nanobody were analyzed by SDS-PAGE, and nanobody-containing fractions (>90% purity) were stored at -20 °C.
4
Purification of Recombinant LTBP-2 and -4S
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Purification of recombinant LTBP-2 and -4S with a FLAG and a 6x His tag was described previously19 (link)26 (link). Briefly, these proteins were purified using TALON affinity resin (Takara) from serum-free conditioned medium of 293T cells stably transfected with pEF6/FLAG-LTBP-2 or -4S.
5
Large-scale E3 Purification via T7 Shuffle
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For large scale purifications of E3, the plasmid was isolated and used to transform T7 shuffle bacteria. An overnight culture was used to inoculate fresh media which was grown at 37°C until a OD600 of ~1.2 was reached. At this point the temperature was lowered to 18°C, 1mM IPTG was added, and protein expression was continued overnight. Cells were harvested by centrifugation and lysed with an Emulsiflex. Nanobodies were purified using Talon affinity resin (Takara Bio, Ann Arbor, MI). Aliquots of E3 were chemically biotinylated using NHS-Biotin (Thermo Fisher, Waltham, MA).
6
Purification and Antibody Generation for OSH1 Protein
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Polyclonal antisera that recognize OSH1 protein were raised against a bacterially expressed histidine-tagged OSH1 fusion protein. The fusion protein was solubilized in a buffer containing urea (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 8 M urea) and then purified with TALON Affinity Resin (Takara Bio). The purified histidine-tagged OSH1 fusion protein was used to raise OSH1 polyclonal antisera in rabbits. IgG that recognizes OSH1 protein was purified by using histidine-tagged OSH1 fusion protein blotted onto PVDF membrane (Immobilon-P, filter type: PVDF, pore size: 0.45 μm; Millipore) followed by washing, elution (0.1 M glycine-HCl, pH 3.0, 0.15 M NaCl), and neutralization (0.5 M HEPES-KOH, pH 8.5).
7
Heterologous Expression and Purification of Neisseria gonorrhoeae NHBA
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Escherichia coli BL21(DE3) was transformed with pET19b carrying the mature NHBA (no signal sequence) from N. gonorrhoeae 1291 (amplified using 5’-ATTActcgagTCGCCCGATGTCAAGTC-3’ and 5’-TGAAggatccCGGCATCAACATCAATC-3’ primers containing XhoI and BamHI sites shown in lower case, in the respective primers). The expression of recombinant NHBA (rNHBA) was induced (100 mM isopropyl β-d-1-thiogalactopyranoside [IPTC], 16 hours, 25°C) and protein-purified using TALON affinity resin (Clontech), as described previously [40 ].
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Escherichia coli BL21(DE3) was transformed with pET19b carrying the mature NHBA (no signal sequence) from N. gonorrhoeae 1291 (amplified using 5’-ATTActcgagTCGCCCGATGTCAAGTC-3’ and 5’-TGAAggatccCGGCATCAACATCAATC-3’ primers containing XhoI and BamHI sites shown in lower case, in the respective primers). The expression of recombinant NHBA (rNHBA) was induced (100 mM isopropyl β-d-1-thiogalactopyranoside [IPTC], 16 hours, 25°C) and protein-purified using TALON affinity resin (Clontech), as described previously [40 ].
8
Cloning and Expression of Macrodomain Proteins
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Human full length MacroD1 was amplified from a human HeLa cDNA library and cloned into pDONR221 (Thermo Fisher) entry vector and a N-terminal truncation mutant was generated the same way by excluding the first 77 amino acids using a different N-terminal primer. SCO6450 (UniProt: Q9ZBG3) was cloned from total Streptomyces coelicolor DNA. For transient transfection in human cells, full-length and truncated MacroD1 pDONR221 vectors were recombined using the Gateway LR reaction (Thermo Fisher) into the pDEST47 destination vector for the expression of C-terminal GFP fused proteins in human cell lines. PARP1 EQ was expressed in pET28 vector and was purified as previously described (Sharifi et al., 2013 (link)). DarT was expressed in pBAD vector, transformed into BL21 strains, induced with arabinose and purified using TALON affinity resin (Clontech) as previously described (Jankevicius et al., 2016 (link)). Macrodomain proteins were expressed in pDEST17 or pET15b and purified as previously described (Chen et al., 2011 (link)).
9
Purification and Characterization of Mycobacterial Kinases
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N-terminally his-MBP-tagged kinase domains of the nine canonical serine-threonine protein kinases from Mtb were expressed and purified as described (Kieser et al., 2015 (link)). His-CwlM was expressed with 1 mM IPTG at 14° for 40 hr in ArcticExpress DE3 RP (Agilent, Lexington, MA). Cells were resuspended and French-pressed in Ni Wash Buffer (50 mM NaHPO4 pH 8.0, 300 mM NaCl, 20 mM imidazole), and supernatants were poured over TALON affinity resin (Clontech, Mountain View, CA). Bound proteins were washed and eluted with Ni Wash Buffer + 200 mM imidazole. Soluble proteins were separated from aggregates on a Superdex S200 gel filtration column (GE Healthcare, Westborough, MA) in 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT. Soluble proteins were concentrated and stored in 50 mM NaPO4 pH 7.5, 150 mM NaCl, 20% glycerol, 2 mM DTT, 1 mM PMSF. Kinase reactions, α-phospho-threonine western blots and mass spectrometry were performed as described (Kieser et al., 2015 (link)).
10
Purification of Recombinant Protein from E. coli
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E. coli M15 cells were transformed with this construct and grown at 37°C in LB medium containing ampicillin and kanamycin. The cells were grown till 0.6 (O.D.600nm) and the expression of the protein was induced with 0.5 mM IPTG for 3 hours. The cells were harvested and stored at -70°C. The cells were subsequently resuspended in lysis buffer (10 mM NaH2PO4, 100 mM NaCl, pH7.0, 1 mM PMSF (Sigma, St. Louis, MO), 1X Halt Protease inhibitor cocktail (ThermoFisher Scientific, Waltham, MA) and lysed by passing through the French press at 20,000 psi. Cell lysate was centrifuged at 20,000×g for 15 minutes at 4°C. The supernatant was collected and again centrifuged at 100,000×g for 1 hour. Cell homogenate was applied onto TALON affinity resin (Clontech, Mountain View, CA) pre-equilibrated with lysis buffer. Resin was washed with five volumes of lysis buffer containing 20 mM imidazole. Bound protein was eluted with 150 mM imidazole containing lysis buffer. Imidazole was then removed by dialysis; the buffer was exchanged with 10 mM NaH2PO4, 100 mM NaCl, pH 6.1 and the protein was concentrated in sucrose bed. Commercially synthesized and HPLC-purified (> 95%) KH16p and KH16 peptides were purchased from GL Biochem (Shanghai, China) and used as received.
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E. coli M15 cells were transformed with this construct and grown at 37°C in LB medium containing ampicillin and kanamycin. The cells were grown till 0.6 (O.D.600nm) and the expression of the protein was induced with 0.5 mM IPTG for 3 hours. The cells were harvested and stored at -70°C. The cells were subsequently resuspended in lysis buffer (10 mM NaH2PO4, 100 mM NaCl, pH7.0, 1 mM PMSF (Sigma, St. Louis, MO), 1X Halt Protease inhibitor cocktail (ThermoFisher Scientific, Waltham, MA) and lysed by passing through the French press at 20,000 psi. Cell lysate was centrifuged at 20,000×g for 15 minutes at 4°C. The supernatant was collected and again centrifuged at 100,000×g for 1 hour. Cell homogenate was applied onto TALON affinity resin (Clontech, Mountain View, CA) pre-equilibrated with lysis buffer. Resin was washed with five volumes of lysis buffer containing 20 mM imidazole. Bound protein was eluted with 150 mM imidazole containing lysis buffer. Imidazole was then removed by dialysis; the buffer was exchanged with 10 mM NaH2PO4, 100 mM NaCl, pH 6.1 and the protein was concentrated in sucrose bed. Commercially synthesized and HPLC-purified (> 95%) KH16p and KH16 peptides were purchased from GL Biochem (Shanghai, China) and used as received.
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