The expression plasmid for human GNA-1 with N-terminal His6-tag was cloned previously40 (link). Human GNA-1 with N-terminal His6-tag was expressed in Rosetta (DE3) E. coli cells. LB cultures were incubated at 37 °C until an OD600 of 0.4–0.6 was reached. Protein expression was induced with 0.5 mM IPTG for 3 h at 37 °C. Harvested pellets were stored at −80 °C. GNA-1 purification protocol was adopted from Hurtado-Guerrero et al.67 (link) with small modifications. E. coli were lysed in 50 mM HEPES/NaOH pH 7.2, 500 mM NaCl, 10 mM imidazole, 2 mM 2-mercaptoethanol, 5% (v/v) glycerol with complete EDTA-free protease inhibitor cocktail (Roche), and 10 µg/ml DNAseI (Sigma) by sonication. The lysate was clarified by centrifugation and the supernatant loaded on Ni-NTA Superflow affinity resin (Qiagen). The resin was washed with wash buffer (50 mM HEPES/NaOH pH 7.2, 500 mM NaCl, 50 mM imidazole, 5% (v/v) glycerol) and the protein was eluted with wash buffer containing 250 mM imidazole. Eluted protein was then dialyzed against storage buffer (20 mM HEPES/NaOH pH 7.2, 500 mM NaCl, 5% (v/v) glycerol).
Ni nta superflow affinity resin
Manufactured by Qiagen
Sourced in Spain
Ni-NTA Superflow affinity resin is a nickel-charged, high-flow agarose-based resin designed for the purification of His-tagged proteins. It utilizes the high affinity interaction between nickel ions and the histidine tag to selectively capture and purify the target proteins.
Automatically generated - may contain errors
8 protocols using ni nta superflow affinity resin
1
Recombinant Human GNA-1 Protein Purification
2
Purification of His-tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sf21 cells were lysed by sonication in lysis buffer (50 mM Tris/HCl pH 7.5, 200 mM NaCl, 10 mM Imidazole, 2 mM TCEP, 0.5 mM Na2Frc6P, 10% (v/v) glycerol) supplemented with complete EDTA-free protease inhibitor cocktail (Roche) and 10 µg/ml DNAseI (Sigma-Aldrich). Cell debris and protein aggregates were removed by centrifugation and the supernatant was loaded on a Ni-NTA Superflow affinity resin (QIAGEN). The resin was washed with lysis buffer and the protein eluted with lysis buffer containing 200 mM imidazole. The proteins were further purified according to their size on a HiLoad 16/60 Superdex 200 prep grade prepacked column (GE Healthcare) using an ÄKTAprime Chromatography System at 4°C with an SEC buffer containing 50 mM Tris/HCl, pH 7.5, 2 mM TCEP, 0.5 mM Na2Frc6P, and 10% (v/v) glycerol.
3
Recombinant Human GNA-1 Protein Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human GNA-1 with N-terminal His6-tag was expressed in Rosetta (DE3) E. coli cells. LB cultures were incubated at 37 °C and 180 r.p.m. until an OD600 of 0.4–0.6 was reached. Then, protein expression was induced by addition of 0.5 mM isopropyl-β-d -1-thiogalactopyranosid and incubated for 3 h at 37 °C and 180 r.p.m. Cultures were harvested and pellets stored at −80 °C. Human GNA-1 purification protocol was adopted from Hurtado-Guerrero et al.59 (link) with small modifications. E. coli were lysed in 50 mM HEPES/NaOH pH 7.2, 500 mM NaCl, 10 mM Imidazole, 2 mM 2-mercaptoethanol, 5% (v/v) glycerol with complete EDTA-free protease inhibitor cocktail (Roche), and 10 µg/ml DNAseI (Sigma) by sonication. The lysate was clarified by centrifugation and the supernatant loaded on Ni-NTA Superflow affinity resin (Qiagen). The resin was washed with wash buffer (50 mM HEPES/NaOH pH 7.2, 500 mM NaCl, 50 mM Imidazole, and 5% (v/v) glycerol), and the protein was eluted with wash buffer containing 250 mM imidazole. Eluted protein was then dialyzed against storage buffer (20 mM HEPES/NaOH pH 7.2, 500 mM NaCl, 5% (v/v) glycerol).
4
Expression and Purification of His-Tagged Human GNA1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression plasmid for human GNA1 with N-terminal His6-tag was cloned previously (Ruegenberg et al., 2020 (link)). Human GNA1 with N-terminal His6-tag was expressed in Rosetta (DE3) E. coli cells. LB cultures were incubated at 37°C and 180 rpm until an OD600 of 0.4–0.6 was reached. Then, protein expression was induced by addition of 0.5 mM IPTG and incubated for 3 hr at 37°C and 180 rpm. Cultures were harvested and pellets were stored at –80°C. Human GNA1 purification protocol was adopted from Hurtado-Guerrero et al., 2008 (link) with small modifications. E. coli were lysed in 50 mM HEPES/NaOH pH 7.2, 500 mM NaCl, 10 mM imidazole, 2 mM 2-mercaptoethanol, 5% (v/v) glycerol with complete EDTA-free protease inhibitor cocktail (Roche), and 10 µg/ml DNAseI (Sigma-Aldrich) by sonication. The lysate was clarified by centrifugation and the supernatant was loaded on Ni-NTA Superflow affinity resin (Qiagen). The resin was washed with wash buffer (50 mM HEPES/NaOH pH 7.2, 500 mM NaCl, 50 mM imidazole, and 5% (v/v) glycerol) and the protein was eluted with wash buffer containing 250 mM imidazole. Eluted protein was then dialyzed against storage buffer (20 mM HEPES/NaOH pH 7.2, 500 mM NaCl, and 5% (v/v) glycerol).
5
Purification of Oxygen-Evolving PSII Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wild-type cells of Synechocystis 6803 containing a single psbA gene (psbA2) and containing a hexahistidine-tag fused to the C terminus of CP47 (73 (link)) were propagated as described previously (40 (link), 74 (link)). Oxygen-evolving PSII core complexes were purified with Ni-NTA superflow affinity resin (Qiagen, Inc., Valencia) at 4 °C under dim green light as described previously (74 (link)). Purified PSII core complexes (in 1.2 M betaine, 10% [vol/vol] glycerol, 50 mM MES-NaOH [pH 6.0], 20 mM CaCl2, 5 mM MgCl2, 50 mM histidine, 1 mM EDTA, and 0.03% [wt/vol] β-DM) were concentrated via ultrafiltration to 1 mg of Chl/mL, aliquoted, frozen in liquid N2, and stored at −80 °C. To prepare samples for cryo-EM analysis, aliquots (50 μg of Chl a) were exchanged into cryo-EM buffer (0.5 M betaine, 50 mM MES-NaOH [pH 6.8], 20 mM CaCl2, 5 mM MgCl2, 0.02% [wt/vol] β-DM) by passage through Bio-Rad Micro Bio-Spin 6 centrifugal gel filtration columns (Bio-Rad Laboratories, Hercules) at 50 × g and then concentrated to ∼2 mg of Chl/mL with Amicon Ultra 0.5-mL 100-kDa centrifugal filter devices (EMD Millipore, Billerica). Samples were maintained at ∼4 °C until being applied to cryo-EM grids. After being exchanged into cryo-EM buffer, samples exhibited light-saturated O2 evolution activities of 5,300 ± 200 µmol O2 (mg of Chl)−1/hr−1 as measured (75 (link)) with a Clark-type oxygen electrode.
6
Purification and Crystallization of Biological Macromolecules
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
2′-Deoxyribonucleotide 5′-triphosphates were obtained from New England Biolabs. 3′-Amino-2′,3′-dideoxyribonucleotide 5′-triphosphates were obtained from Trilink Biotechnologies. DNA oligonucleotides were purchased from IDT. Oligonucleotides incorporating a 3′-amino residue were synthesized and purified by standard methods, using phosphoramidites obtained from Chemgenes or as recently reported (13 (link)) and using reagents purchased from Glen Research on an Expedite 8909 synthesizer. Premixed reagent solutions for crystallization were purchased from Hampton Research. Recombinant lysozyme and Bugbuster lysis reagent were purchased from EMD. Ni-NTA Superflow affinity resin was purchased from Qiagen. All other buffers and reagents were prepared from high-purity chemicals obtained from Sigma-Aldrich, using chemicals with trace metals analysis where available. Competent cells were obtained from New England Biolabs.
7
Purification of Recombinant GFAT-1 and Isomerase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sf21 cells (full-length GFAT-1) or E. coli (isomerase domain) were lysed by sonication in lysis buffer (50 mM Tris/HCl pH 7.5, 200 mM NaCl, 10 mM Imidazole, 2 mM Tris(2-carboxyethyl)phosphin (TCEP), 0.5 mM Na2Frc6P, 10% (v/v) glycerol, supplemented with complete EDTA-free protease inhibitor cocktail (Roche), and 10 µg/ml DNAseI (Sigma)). Samples were cleared by centrifugation and the supernatant was loaded on Ni-NTA Superflow affinity resin (Qiagen). The resin was washed with lysis buffer and protein was eluted with lysis buffer containing 200 mM imidazole. For the isomerase domain, the His6-tag was proteolytically removed using 5 U of thrombin (Sigma-Aldrich) per mg protein overnight at 4 °C, followed by an additional IMAC purification. Full-length GFAT-1 and the isomerase domain were further purified according to their size on a HiLoad™ 16/60 Superdex™ 200 prep grade prepacked column (GE Healthcare) using an ÄKTAprime chromatography system at 4 °C with a SEC buffer containing 50 mM Tris/HCl, pH 7.5, 2 mM TCEP, 0.5 mM Na2Frc6P, and 10% (v/v) glycerol.
8
Purification of GFAT-1 Protein Complex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sf21 cells were lysed by sonication in lysis buffer (50 mM Tris/HCl pH 7.5, 200 mM NaCl, 10 mM Imidazole, 2 mM tris(2-carboxyethyl)phosphin (TCEP), 0.5 mM Na2Frc6P, 10% (v/v) glycerol, supplemented with complete EDTA-free protease inhibitor cocktail (Roche) and 10 µg/ml DNAseI (Sigma)). Cell debris and protein aggregates were removed by centrifugation and the supernatant was loaded on an Ni-NTA Superflow affinity resin (Qiagen). The resin was washed with lysis buffer and the protein eluted with lysis buffer containing 200 mM Imidazole. Subsequently, proteins were separated according to their size on a HiLoad™ 16/60 Superdex™ 200 prep grade prepacked column (GE Healthcare) using an ÄKTAprime chromatography system at 4 °C with a SEC buffer containing 50 mM Tris/HCl, pH 7.5, 2 mM TCEP, 0.5 mM Na2Frc6P, and 10% (v/v) glycerol. For co-crystallization of GFAT-1 with GlcN6P, GFAT-1 was purified in the absence of 0.5 mM Na2Frc6P.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!