For expression of GmFULa protein in bacteria, the GmFULa full-length coding sequences were inserted into the inducible expression vector pET-32a (with 6 × His Tag) between BamHI and SacI sites. The resulting plasmids were transformed into Escherichia coli strain Rosetta (DE3) and induced using 0.4 mM isopropyl-b-ß-1-thiogalactopyranoside (IPTG) at 25 °C for 12 h. The recombinant protein was purified using ProteinlsoR Ni–NTA Resin (Transgen biotech, Beijing, China) according to the manufacturer’s protocol. For GmSUSs and GmSUTs, the probe fragment consisted of a region of 40 bp with the canonical CArG box (C[A/T]8G) in the center (Table S1 for primer sequences). The mutated CArG box fragment TM was CCGCG (AATAT) in the mid region of the TG motif. The probes were labeled using the EMSA Probe Biotin Labeling Kit (Beyotime Biotechnology). The same fragments without biotin labeling were used as competitors. The protein–DNA complexes were separated with 6% native polyacrylamide gels. The Biotin-labeled probes were visualized using Chemiluminescent Biotin-labeled Nucleic Acid Detection Kit (Beyotime Biotechnology) according to the manufacturer’s protocol.
Proteinlsor ni nta resin
Manufactured by Transgene
Sourced in China
The ProteinlsoR Ni–NTA Resin is a nickel-based affinity chromatography resin used for the purification of histidine-tagged recombinant proteins. It utilizes the high affinity between nickel ions and histidine residues to capture and isolate the target proteins from complex samples.
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2 protocols using proteinlsor ni nta resin
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Recombinant Expression and Purification of GmFULa Protein
2
GmFULa Protein Expression in Bacteria
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For expression of GmFULa protein in bacteria, the GmFULa full-length coding sequences were inserted into the inducible expression vector pET-32a (with 6 × His Tag) between BamHI and SacI sites. The resulting plasmids were transformed into
Escherichia coli strain Rosetta (DE3) and induced using 0.4 mM isopropyl-b-ß-1thiogalactopyranoside (IPTG) at 25°C for 12h. The recombinant protein was purified using Proteinlso R Ni-NTA Resin (Transgen biotech, Beijing, China) according to the manufacturer's protocol. For GmSUSs and GmSUTs, the probe fragment consisted of a region of 40 bp with the canonical CArG box (C[A/T] 8 G) in the center (Table S1 for primer sequences). The mutated CArG box fragment TM was CCGCG (AATAT) in the mid region of the TG motif. The probes were labeled using the EMSA Probe Biotin
Labeling Kit (Beyotime Biotechnology). The same fragments without biotin labeling were used as competitors. The protein-DNA complexes were separated with 6% native polyacrylamide gels. The Biotin-labeled probes were visualized using Chemiluminescent Biotin-labeled Nucleic Acid Detection Kit (Beyotime Biotechnology) according to the manufacturer's protocol.
Escherichia coli strain Rosetta (DE3) and induced using 0.4 mM isopropyl-b-ß-1thiogalactopyranoside (IPTG) at 25°C for 12h. The recombinant protein was purified using Proteinlso R Ni-NTA Resin (Transgen biotech, Beijing, China) according to the manufacturer's protocol. For GmSUSs and GmSUTs, the probe fragment consisted of a region of 40 bp with the canonical CArG box (C[A/T] 8 G) in the center (Table S1 for primer sequences). The mutated CArG box fragment TM was CCGCG (AATAT) in the mid region of the TG motif. The probes were labeled using the EMSA Probe Biotin
Labeling Kit (Beyotime Biotechnology). The same fragments without biotin labeling were used as competitors. The protein-DNA complexes were separated with 6% native polyacrylamide gels. The Biotin-labeled probes were visualized using Chemiluminescent Biotin-labeled Nucleic Acid Detection Kit (Beyotime Biotechnology) according to the manufacturer's protocol.
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