The SBP‐domain of OsSPL4 (SPL4SBP) encoded by the sequences from 193 to 426 bp was amplified using PCR with the primers SPL4SBP‐F/R. Then, the amplified fragment was purified and inserted into the pGEX‐6p‐1 vector to express a glutathione S‐transferase fusion protein (GST‐SPL4SBP) in Transetta cells (DE3; Transgene Biotech, Beijing, China). The proteins were purified via glutathione sepharose 4B beads (GE Healthcare, Chicago, IL, USA). Oligonucleotide probes containing GTAC motifs were synthesized and labelled (GH3.2pro‐EMSA‐BiotinF/R) with biotin at the 5′ end, or without biotin (GH3.2pro‐EMSA‐F/R), by Sangon Biotech. The probe primers are listed in Table S1 . The binding reaction contained purified GST‐SPL4SBP, EMSA/Gel‐Shift binding buffer and probes. GST protein alone was used as a negative control. EMSA assay was conducted with the Chemiluminescent EMSA Kit (Beyotime) in accordance with the manufacturer’s instructions. The detailed EMSA procedure also followed the manufacturer's instructions. Images were captured using a charge‐coupled device camera (ChemiDoc XRS+; Bio‐Rad).
Transetta de3 cells
Manufactured by Transgene
Sourced in China
Transetta (DE3) cells are a strain of Escherichia coli (E. coli) bacteria that are commonly used in molecular biology and protein expression experiments. These cells contain a chromosomally integrated T7 RNA polymerase gene, which is induced by the presence of the chemical compound isopropyl β-d-1-thiogalactopyranoside (IPTG). This allows for the high-level expression of recombinant proteins under the control of a T7 promoter.
Automatically generated - may contain errors
Lab products found in correlation
Glutathione sepharose 4b beads, by GE Healthcare (2 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Chemiluminescent emsa kit, by Beyotime (1 mentions)
Pgex 4t 1 vector, by GE Healthcare (1 mentions)
Chemiluminescent emsa kit, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose 4b beads, by Solarbio (1 mentions)
Amicon ultra 15 filter, by Merck Group (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
0.45 μm filter, by Merck Group (1 mentions)
Bamhi xhoi, by Takara Bio (1 mentions)
Axyprep dna gel extraction kit, by Corning (1 mentions)
Axyprep plasmid miniprep kit, by Corning (1 mentions)
T4 ligase, by Takara Bio (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Sangon (1 mentions)
Trans t1 cells, by Transgene (1 mentions)
Bam hi sal 1, by Takara Bio (1 mentions)
Pgem t easy vector, by Transgene (1 mentions)
7 protocols using transetta de3 cells
1
Characterization of OsSPL4 SBP Domain
2
Characterization of SPL9-miR528-AO Pathway
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The DNA-binding domain of SPL9 (SPL9 SBP) was amplified using PCR, then inserted into the pGEX4T1 vector (GE Healthcare, Chicago, IL, USA) and expressed as a glutathione S-transferase fusion (C) A model of the SPL9-miR528-AO pathway regulating plant antiviral immunity. SPL9 binds to the GTAC motif in the miR528 promoter to activate its transcription. MiR528 is loaded onto AGO1 and forms an RNA-induced silencing complex to cleave the target, AO, and thus regulates the rice response to RSV infection. protein (SPL9 SBP-GST) in Transetta cells (DE3; TransGen Biotech, Beijing, China). The fusion proteins were purified using glutathione Sepharose 4B beads (GE Healthcare). The primers used for the amplification of SPL9 SBP are listed in Supplemental Data 2. Oligonucleotide probes containing GTAC motifs were synthesized and labeled with biotin (Ruibio, Beijing, China). An EMSA was performed using the Chemiluminescent EMSA Kit (Thermo Fisher Scientific). The probe sequences are shown in Supplemental Data 2.
3
GRP78 Protein Interaction Studies
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Rat GRP78-full length (FL) (28–654), GRP78-NBD (28–405), GRP78-SBD (422–654) proteins were cloned into pGEX-6p-1 vector containing a GST-tag. All constructs were expressed in Transetta (DE3) cells (TransGen Biotech, Beijing, China), and were grown at 37 °C for 5 h, then 0.8 mM IPTG was added at 37 °C for 3 h followed by 20 ℃ for 24 h and then purified with glutathione Sepharose 4B beads (Solarbio biotechnology Co., Ltd, Beijing, China). For overexpression of Flag-VEGF-B in the 293T cells, cells were transfected with 20 μg of Flag-VEGF-B per plate. Transient transfection was performed using Lipofectamine 2000 (Thermo, USA) according to the manufacturer’s instructions. The cells were then collected at 30 h post transfection and lysed at 4 °C. Flag-VEGF-B protein was rotated with GST-GRP78-FL, GST-GRP78-NBD, GST-GRP78-SBD at 4 °C for 4 h. After centrifugation and three washes, the beads were eluted with 50 μl of 2 × SDS-PAGE loading buffer and then boiled for 10 min, followed by western blot.
4
Recombinant Expression and Characterization of IbSSI
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The ORF of IbSSI was amplified using the ES-F/R primers and inserted into the pET-28a vector. Next, the sequence-validated pET-28a-IbSSI vector and native pET-28a vector were introduced into competent E. coli strain Transetta (DE3) cells (Transgen Biotech, Beijing, China). Positive clones were grown in Luria-Bertani (LB) medium. Fresh LB medium was inoculated with the overnight cultures at a 100:1 (v:v) dilution. Soluble cytoplasmic proteins were prepared from isopropyl β-D-thiogalactopyranoside (IPTG)-induced Transetta (DE3) cells59 (link). The expressed IbSSI protein was subjected to SDS-PAGE analysis according to the method of Jiang et al.60 (link), and a starch synthase assay was conducted as described previously61 (link). One unit of activity was defined as the formation of 1 nmol of ADP per min at 30 °C.
5
Purification of SARS-CoV-2 Nsp13 Helicase
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pET-28a-nsp13 and pET-28a-nsp13-K289A were transformed into Transetta (DE3) cells (TransGen Biotech, China), grown at 37℃, and induced with IPTG (0.2 mM) when the optical density reached ∼0.8. Thereafter, the induced cells were grown at 18℃ for 16 h. Cells were harvested at 15,000×g by centrifugation at 4℃. After ultrahigh-pressure disruption and centrifugation at 20,000×g, the supernatants were filtered through a 0.45-μM filter (Millipore, MA, USA), then run through a Ni-affinity column. The proteins were then eluted with a linear-gradient concentration of imidazole from 20 to 250 mM. The eluates were concentrated, and the buffers were replaced with Buffer B2 (0.2 M Tris−HCl, 200 mM NaCl) using Amicon Ultra-15 filters (Millipore). The concentration of purified protein was determined using an enhanced BCA protein assay kit (Beyotime, China).
6
Recombinant Expression and Purification of GmIFR
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The full-length cDNA of GmIFR was fused to the N-terminus of the 6 × His-tag, at the NcoI and XhoI restriction sites of the vector pET28a (+) (Novagen, Germany). The recombinant fusion plasmid was expressed into Transetta (DE3) cells (TransGen Biotech, China). His-tagged proteins were induced with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) at 37°C for 4 h. The fusion protein was purified at room temperature and quantified according to the pET System Manual (Novagen). The fusion GmIFR protein was subsequently analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting using anti-His antibody.
7
Cloning and Expression of Karyopherin Proteins
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Open reading frames (ORFs) of the Bm-KPNAs and Bm-KPNB1 were cloned in a pGEM ® -T Easy Vector (Transgen) and amplified in Trans-T1 cells (Transgen). Plasmids were extracted using an AxyPrep TM Plasmid Miniprep Kit (Axygen, Suzhou, China). Bm-KPNA1, Bm-KPNA3, and Bm-KPNB1 were double-digested by BamHI/ XhoI (Takara), and KPNA2 was digested by BamHI/ SalI (Takara) at 37°C for 4 h. The prokaryote expression plasmid pET-28a+ was also doubledigested by the above enzyme pairs, under similar conditions. Target fragments were collected by agarose electrophoresis and purified using an AxyPrep TM DNA Gel Extraction Kit (Axygen). The targeted gene fragments and pET-28a+ were linked by T4 ligase (Takara) at 25°C for 1 h. The recombinant plasmids were verified by DNA sequencing and transferred into Transetta (DE3) cells (Transgen). Recombinant protein expression was induced by different concentrations of isopropyl β-d-1-thiogalactopyranoside (IPTG) (Sangon, Shanghai, China) at 30°C for 4 h.
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