For autophosphorylation analyses in vitro, the cytoplasmic domain (824 to 1207 aa) of SlBRI1, S1040A, S1040D, or K916E was amplified from PSlBRI1::SlBRI1-GFP-pBI121, PSlBRI1::S1040A-GFP-pBI121, PSlBRI1::S1040D-GFP-pBI121, or PSlBRI1::SlBRI1-GFP-pBI121 with the specific primers (SlBRI1-CD-F and SlBRI1-CD-R) listed in Table S1 . The amplified fragment was subcloned into the prokaryotic expression vector pFLAG-MAC and then transformed into E. coli BL21 (DE3) pLysS (Transgene, CD901-02, Beijing, China). SlBRI1-FLAG-MAC and K916E-FLAG-MAC were used as the positive and negative controls, respectively, and subsequent protein purification and autophosphorylation analyses in vitro were performed according to previously described methods [21 (link),53 (link)]. Intensities of bands were quantified by using ImageJ software (NIH, Bethesda, Maryland, USA) and presented as relative values compared with the FLAG-SlBRI1.
Bl21 de3 plyss
Manufactured by Transgene
Sourced in China
BL21 (DE3) pLysS is a bacterial expression strain commonly used for the production of recombinant proteins. It is derived from the E. coli BL21 (DE3) strain and contains the pLysS plasmid. The pLysS plasmid provides low-level expression of T7 lysozyme, which helps to reduce basal expression of the target protein prior to induction. This strain can be used for the expression of a wide range of recombinant proteins.
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Lab products found in correlation
Nadph, by Roche (1 mentions)
Tryptone, by Sangon (1 mentions)
Yeast extract, by Sangon (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Plasmid purification kit, by Qiagen (1 mentions)
Kod plus mutagenesis kit, by Toyobo (1 mentions)
Xanthine oxidase, by Merck Group (1 mentions)
Xanthine, by Merck Group (1 mentions)
Nickel nitrilotriacetic acid resin, by Qiagen (1 mentions)
Sephadex g 25 resin, by Qiagen (1 mentions)
Pfu dna polymerase, by New England Biolabs (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
Nickel nitrilotriacetic affinity chromatography, by GE Healthcare (1 mentions)
Ni sepharose 6 fast flow, by GE Healthcare (1 mentions)
Prset a vector, by Thermo Fisher Scientific (1 mentions)
8 protocols using bl21 de3 plyss
1
In Vitro BRI1 Autophosphorylation Assay
2
Heterologous Expression of AspDH Enzymes
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AspDH genes from K. pneumoniae 34618, Delftia sp. Cs1–4, Methanosphaerula palustris, Roseibacterium elongatum, Bradyrhizobium japonicum and Ralstonia eutropha JMP134 were synthesized by Genewiz (Suzhou, China) with codon optimization. E. coli BL21(DE3) pLysS (Transgen) was used for heterologous expression. The plasmid pED31 was used for the expression of AspDH. NAD+, NADH, NADPH, and NADP+ were purchased from Roche. All other chemicals used in this work were of analytical grade and commercially available.
3
Plasmid-based Recombinant Protein Production
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Plasmid pET22 was used as the vector for gene cloning and expression. E. coli strain DH5α (Transgen, China) was used as the host for cloning, and E. coli strain BL21 (DE3) plysS (Transgen, China) was used as protein expression. E. coli cells were grown at 37°C in LB medium containing 10g NaCl, 10g tryptone, and 5g yeast extract (Sangon Biotech, China) per liter at pH 7.0, and LB agar medium was added with 1.5–2.0% (w/v) agar.
4
Generation and Characterization of OsASR6 Protein
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For generation of OsASR6 protein, the cDNA sequence of OsASR6 was amplified through using gene-specific primers listed in Supplemental Table S5 , and was then cloned into pET32a and expressed in Escherichia coli BL21 (DE3) pLysS (Trans-Gen Biotech). His-tagged OsASR6 protein was affinity purified and used for the electrophoretic mobility shift assay (EMSA). The biotinylated 4× GGCCCAT, 4× AGCCCAT and 4× GAAAAA probes as well as unlabeled versions of these sequences and mutated sequences were synthesized as forward and reverse strands, and were then renatured to double-stranded probes in annealing buffer [100 mM Tris-HCl, (pH 7.5), 10 mM EDTA, 1 M NaCl] at 100 °C for 5 min. The gel-shift assay was conducted according to the Thermo gel-shift assay system manual.
5
Recombinant Protein Expression and Purification
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Pfu DNA polymerase, T4 DNA ligase, dNTP, and restriction enzymes (Nde I, and Xho I) were purchased from New England Biolabs. The KOD-Plus Mutagenesis Kit was purchased from TOYOBO. The E. coli strains Trans 10 and BL21(DE3) PlysS were obtained from TransGen. The plasmid purification kits, gel extraction kits, nickel nitrilotriacetic acid resin and Sephadex G-25 resin were purchased from QIAGEN (Chatsworth, CA, USA). The SuperdexTM 200 HiLoad 16/60 gel filtration column was from Pharmacia. The xanthine, xanthine oxidase and NBT were obtained from Sigma (St. Louis, MO, USA). The KO2 was purchased from Acros. All other reagents were of analytic grade.
6
Purification of Mutant Ricin Toxin A
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The recombinant plasmid was further transformed into E.coli strain BL21(DE3)pLysS (TransGen Biotech, CD701) and grown in 5 ml LB medium overnight containing 100 μg/ml of ampicillin at 37°C with 200 rpm shaking. The overnight grown culture was inoculated in 500 ml of LB medium and was grown at 37°C in a shaker until it reached an A600 of 0.6. The culture was then induced with 0.4 mM IPTG (Merck, C9H1805S) and the cells were incubated for another 12 h at 20°C for expression of protein containing 6 × His tag at the N-terminal. The induced bacterial cells were harvested and lysed by sonication and centrifuging to separate the supernatant and cell debris. The protein in the supernatant was purified by nickel-nitrilotriacetic affinity chromatography (GE Healthcare, 17–5248) and the concentration was measured by a BCA assay kit (Novagen, 71285–3). As a positive control, the recombinant wild type RTA (rRTA) was prepared and purified using the same methods as the mtRTA, except for the protein inducing condition (medium: TB, inducing temperature: 30°C, IPTG concentration: 0.4 mM, inducing time: 4 h).
7
Bacterial Expression and Purification of Drp1
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For bacterial expression, Drp1 was synthesized and cloned into BamHI between HindIII sites of the pRSETA vector (Thermo Fisher), which includes 6 x His-tag sequence. Escherichia coli BL21 (DE3) plysS (Transgene, Cat No. CD701-02) cells were used for the prokaryotic expression of Drp1 protein. Cells were grown in Luria-Bertani (LB) media containing 100 μg/ml ampicillin at 37 °C until the OD600 of cell density was 0.5. 0.1 mmol/L isopropyl 1-thio-β-d -galactopyranoside was added to induce protein expression and the temperature was shifted to 4 °C for 7 days. Bacteria were harvested, suspended in 50 mmol/L potassium phosphate buffer (pH = 7.4) and lysed via ultrasonication. After centrifuged at 12,000 rpm for 30 min at 4 °C, the His-tagged Drp1 was purified by Nickle resin (Ni Sepharose 6 Fast Flow, GE Healthcare) followed by 300 mmol/L imidazole elution, desalted and exchanged into 100 mmol/L HEPES buffer with Sephadex column. Drp1 protein was diluted to the required concentration before assay.
8
Cultivation and Characterization of H. pylori
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The H. pylori strain used in this study was ATCC 26695 stored at -80°C in the laboratory. The strain was streaked onto the Karmali agar plate supplemented with Karmali Agar base (CM 0935, Oxoid) containing 15% defibrinated sheep blood, and the plate was incubated at 37°C under microaerobic conditions (5% O2, 10% CO2, and 85% N2) for 3-5 days. The obtained colony was confirmed by urease, oxidase, and catalase traits and inspection of bacterial morphology. The Escherichia coli (E. coli) strains DH5α and BL21 (DE3) pLysS (Transgen Biotech, Beijing, China) used as the host strain for molecular cloning and protein expression were grown on Luria-Bertani plates (LB, Land Bridge, Beijing, China) for 18-24 h at 37°C with appropriate antibiotics.
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