MjRPS27 exF and MjRPS27 exR (Table 1 ) were used to amplify MjRPS27 by RT-PCR. The PCR product and empty plasmid pGEX4T-2 (GE Healthcare) were digested by two restriction endonucleases, BamHI and XhoI (Thermo Scientific), at 37°C for 1 h (MjRPS27) and 37°C for 0.5 h (vector pGEX4T-2). The obtained fragments and the pGEX4T-2 plasmids ligated with T4 DNA ligase (Thermo Fisher) to construct the recombinant plasmid pGEX4T-2/MjRPS27. The constructed recombinant plasmid was then transformed into E. coli DH5α cells, cultured at 37°C overnight. The recombinant plasmid, purified from the E. coli DH5α, was transformed into E. coli Rosetta cells, and MjRPS27 expression was induced with β-d -1-thiogalactopyranoside (IPTG; Sangon, Shanghai, China) at a final concentration of 0.5 mM at 37°C. Rosetta bacteria were collected and disrupted with ultrasonic waves. The crushed bacterial solution was centrifuged at 12,000 × g for 10 min at 4°C, and the supernatant and precipitate were collected and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The recombinant protein was purified by GST-resin chromatography (GenScript, Nanjing, China) following the manufacturer's instructions.
Pgex 4t 2
Manufactured by Thermo Fisher Scientific
The PGEX-4T-2 is a plasmid vector designed for the expression of fusion proteins in Escherichia coli. It features a tac promoter for high-level inducible expression, a polylinker site for the insertion of the target gene, and a GST tag for purification of the expressed fusion protein.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Pcmv myc vector, by Takara Bio (1 mentions)
Anti myc antibody, by Takara Bio (1 mentions)
Anti rabbit igg conjugated to hrp, by Jackson ImmunoResearch (1 mentions)
Anti mouse igg conjugated to hrp, by Jackson ImmunoResearch (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
T4 ligase, by Takara Bio (1 mentions)
Bradford assay kit, by Sangon (1 mentions)
Restriction enzyme, by Takara Bio (1 mentions)
Ptk787, by MedChemExpress (1 mentions)
Taq dna polymerase, by Takara Bio (1 mentions)
E coli dh5α, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside iptg, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Pan anti propionyl lysine antibody, by PTM Biolabs (1 mentions)
Pgex 4t 2, by GE Healthcare (1 mentions)
Xhoi, by Thermo Fisher Scientific (1 mentions)
Bamhi, by Thermo Fisher Scientific (1 mentions)
4 protocols using pgex 4t 2
1
Cloning and Purification of MjRPS27 Protein
2
Functional Studies of HIV-1 Tat Variants
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TatC (lack Ser46Phe) and Tat variants (TatN12, TatD60, TatVT6) were cloned into: (a) mammalian expression vector pCMV-myc vector (Clonetech) under the CMV promoter for functional studies, and (b) prokaryotic expression vector pGEX-4T-2 (Invitrogen) to obtain GST-tagged proteins. HIV-1 subtype B TAR was cloned in pcDNA3.1 (Invitrogen) for TAR synthesis to determine Tat–TAR binding activity. Anti-Tat antibody (NIH AIDS Reagent Programme), Anti-myc antibody (Clontech), Anti-GAPDH antibody (Cell Signaling Technology), Anti-rabbit IgG conjugated to HRP (Jackson Immunoresearch), and Anti-Mouse IgG conjugated to HRP (Jackson Immunoresearch) were used in western blotting.
3
Pancreatic Cancer Protein Expression Analysis
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The tissue of pancreatic cancer was provided by the Shanghai First Peoples Hospital. Trizol for extracted RNA was obtained from Invitrogen (Shanghai, China). Restriction enzymes, T4 ligase, and Taq DNA Polymerase were purchased from TaKaRa (Dalian, China). The kits for the PCR product cleaning, DNA recovery from the gel after electrophoresis, and plasmid extraction were purchased from Sangon (Shanghai, China). E. coli DH5α from Invitrogen and E. coli BL21 (DE3) from Novagen (Shanghai, China) were used for the plasmid propagation and protein overexpression, respectively. The plasmid pGEX-4T-2 was purchased from Invitrogen. The Bradford assay kit for measuring protein concentrations was purchased from Sangon. The anti-VEGFR-2 antibody and mouse monoclonal antibody against phosphotyrosine (PY99) were purchased from BD (Becton, Dickinson and Company, Shanghai, China). All purified chromatography columns were obtained from GE Healthcare (Shanghai, China). VEGFR-2 expressed in Sf9 (Spodoptera frugiperda) insect cells was purchased from Abcam (Shanghai, China). PTK787, a known VEGFR-2 inhibitor, was obtained from MedChemExpress (Shanghai, China). The isopropyl β-D-1-thiogalactopyranoside (IPTG) and ampicillin were obtained from Sigma-Aldrich (Shanghai, China). All the solutions were made up with MilliQ water.
4
Protein Purification and Characterization from Bacterial Strains
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The strains and plasmids used in this study were listed in Table 2 . Bacillus subtilis 168 and E. coli were grown on Luria-Bertani (LB) medium. When needed, antibiotics were added to the medium at the following concentrations: ampicillin, 100 μg/ml; kanamycin, 50 μg/ml. All media were sterilized by autoclaving at 121 °C for 20 min.
The strains and plasmids used in this study.
| Strain or plasmid | Source or reference |
|---|---|
| strains | |
| Bacillus subtilis 168 | ATCC 6051 |
| E. coli DH5α | TransGen Biotech |
| E. coli BL21(DE3) | TransGen Biotech |
| E. coli BL21(DE3)-BsAcsA | In this work |
| E. coli BL21(DE3)-BsAcuA | In this work |
| Salmonella enterica Δacs (JE7758) | 18 (link) |
| plasmids | |
| pET-28a | Thermo Scientific |
| pBAD30 | 18 (link) |
| pGEX-4T-2 | Thermo Scientific |
| pET-BsAcsA | Lab stock |
| pGEX-BsAcuA | In this work |
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