The gene coding for the Zα domain (140–202) of human ADAR1 (hZαADAR1) was subcloned into pET28a+ (Novagen Inc., WI, USA) and transformed into BL21 (DE3) (Novagen Inc., WI, USA). The cells were grown in a Luria-Bertani medium containing 30 μg/ml kanamycin to an optical density of 0.5–0.6 at 600 nm (OD600) at 37°C. They were induced with 0.5 mM isopropyl β-d -1-thiogalactoside at 37°C for 4 h. hZαADAR1 was purified using methods described previously (17 (link)). After purification in a His-affinity column (GE Healthcare, NJ, USA) and removal of the N-terminal six histidines with thrombin, the protein in buffer A (20 mM HEPES-NaOH, pH 7.5, and 10 mM NaCl) was further purified using Resource S (GE Healthcare, NJ, USA). The purified protein was dialyzed against buffer containing 5 mM HEPES-NaOH (pH 7.5) and 10 mM NaCl. After dialysis, the protein was concentrated to over 1 mM by ultrafiltration on a Centricon-YM3 device (Millipore, MA, USA). All the buffer change steps were carried out using molecular porous membrane tubing (Spectrum Laboratories, Inc., CA, USA). The protein concentration was measured spectroscopically using an extinction coefficient of 6990 M−1cm−1 at 280 nm, calculated at www.expasy.org .
His affinity column
Manufactured by GE Healthcare
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His-affinity column is a chromatography column used for the purification of recombinant proteins containing a histidine tag. The column utilizes the high affinity interaction between the histidine tag and immobilized metal ions, typically nickel or cobalt, to selectively capture and purify the target protein from complex mixtures.
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3 protocols using his affinity column
1
Purification of Human ADAR1 Zα Domain
2
Cloning and Purification of SrtB Mutants
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The DNA sequence of SrtBΔ30 was amplified from S. aureus 29213 genomic DNA as a template and was cloned into pET-28a. After that, the plasmid was transfected into E. coli BL21 (DE3) for expression.
The mutants of SrtBΔ30 (N92A, Y128A) were produced from the pET-28a plasmid using the Quick Change site-directed mutation kit (Stratagene, La Jolla, CA, USA). After digestion with the DpnI enzyme, the plasmids were transfected into E. coli BL21 (DE3). The primer pairs used in this study are presented in TableS1 .
E. coli was cultured in the LuriaBertani (LB) medium. Isopropyl-β-d-thiogalactoside (IPTG) was added when the optical density at 600 nm (OD600) reached 0.8. The bacteria were pelleted by centrifugation and lysed ultrasonically. The supernatant was loaded onto a His-affinity column (GE Healthcare Life Sciences). The contaminant proteins were eliminated with 10 mM imidazole in an equilibration buffer (300 mM NaCl, 50 mM NaH2PO4, 10 mM Tris-HCl, pH 8.0). SrtBΔ30 and the mutant proteins were eluted with 200 mM imidazole in an equilibration buffer.
The mutants of SrtBΔ30 (N92A, Y128A) were produced from the pET-28a plasmid using the Quick Change site-directed mutation kit (Stratagene, La Jolla, CA, USA). After digestion with the DpnI enzyme, the plasmids were transfected into E. coli BL21 (DE3). The primer pairs used in this study are presented in Table
E. coli was cultured in the LuriaBertani (LB) medium. Isopropyl-β-d-thiogalactoside (IPTG) was added when the optical density at 600 nm (OD600) reached 0.8. The bacteria were pelleted by centrifugation and lysed ultrasonically. The supernatant was loaded onto a His-affinity column (GE Healthcare Life Sciences). The contaminant proteins were eliminated with 10 mM imidazole in an equilibration buffer (300 mM NaCl, 50 mM NaH2PO4, 10 mM Tris-HCl, pH 8.0). SrtBΔ30 and the mutant proteins were eluted with 200 mM imidazole in an equilibration buffer.
3
Protein Expression and Purification from Sf9 Cells
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Proteins were expressed and purified as described in ref. 59 (link). Proteins were coexpressed by Baculovirus expression in Sf9 insect cells. Cells were infected at a density of 1 × 106 cells per ml and grown for 72 h. Cells were collected in lysis buffer (50 mM TRIS pH 8, 150 mM NaCl, 2 mM TCEP) and complete EDTA-free protease inhibitor (Sigma) was added. Cells were lysed by sonication and sample was cleared by centrifugation at 21,000 × g for 30 min. Supernatant was loaded on His-affinity column (GE Healthcare) and column was washed with lysis buffer + 50 mM Imidazol. Sample was eluted with 500 mM Imidazol. The eluted sample was then loaded on a Strep-affinity column (IBA Life Science) and eluted using lysis buffer + 2.5 mM desthiobiotin. The sample was then purified on a Superdex 200 size exclusion column (GE Healthcare) in lysis buffer. Samples were concentrated and stored at − 80 °C.
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