The ZIKV NS1 genes were amplified from the cDNA of infected cells and cloned into a pET-28a (+) expression vector (69864, Millipore). The cloning primers are presented in Extended Data Table 1 . Recombinant NS1 proteins were expressed in the Escherichia coli BL21 DE3 strain in an insoluble form in inclusion bodies. The proteins were then solubilized by 8 M urea and purified with a TALON Purification Kit (635515, Clontech). Murine antisera were produced via the inoculation of recombinant ZIKV NS1 with 3 boosts. Polyclonal antibodies were purified from the immunized antisera using Protein A/G agarose (20423, Thermo). The anti-V5-HRP antibody for tag detection were purchased from Invitrogen (R96125, Thermo).
Pet28a expression vector
Manufactured by Merck Group
Sourced in Germany
The PET28a expression vector is a widely used plasmid for the expression of recombinant proteins in Escherichia coli. It features a T7 promoter for high-level protein expression, a kanamycin resistance marker, and a 6xHis tag for purification of the expressed proteins.
Automatically generated - may contain errors
Lab products found in correlation
Talon purification kit, by Takara Bio (3 mentions)
Protein a g agarose, by Thermo Fisher Scientific (2 mentions)
Anti v5 hrp antibody for tag detection, by Thermo Fisher Scientific (2 mentions)
Anti v5 hrp, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Ni nta agarose bead, by Qiagen (1 mentions)
E coli top10f cells, by Thermo Fisher Scientific (1 mentions)
Geneart, by Thermo Fisher Scientific (1 mentions)
Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions)
E coli bl21 de3 strain, by Thermo Fisher Scientific (1 mentions)
E coli krx cells, by Promega (1 mentions)
E coli bl21 ai, by Thermo Fisher Scientific (1 mentions)
14 protocols using pet28a expression vector
1
Recombinant ZIKV NS1 Protein Production
2
Recombinant Japanese Encephalitis Virus Envelope Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The JEV-E gene was amplified from the cDNA of infected cells and inserted into a pET-28a (+) expression vector (Millipore). The cloning primers are presented in Table S1. Recombinant JEV-E protein was expressed in the Escherichia coli BL21 DE3 strain in an insoluble form in inclusion bodies. The protein was then solubilized in 8 M urea and purified with a TALON Purification Kit (Clontech). Murine antiserum was produced via inoculation with recombinant E protein with three boosts, and the serum was divided and stored at −80°C until further use. The samples were separated by 12% SDS-PAGE gel, followed by electrophoretic transfer to polyvinylidene fluoride membranes and blocking and incubating membranes with primary antibodies. The following antibodies were used in these experiments: anti-V5-HRP (Invitrogen) antibodies and anti-GAPDH (Proteintech) antibodies.
3
Purification of NUP153 RNA-Binding Domain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A PCR-amplified N-terminal (amino acids [aa] 235-326) fragment of NUP153 cDNA (NM_005124) was cloned into the EcoRI/SalI site of the pET-28a expression vector (Millipore). The plasmid was transformed into RosettaTM (DE3) Escherichia coli, and the RNA-binding domain (RBD) of NUP153 was purified using Ni-NTA agarose beads (Qiagen, Valencia, CA, USA). Bead-bound protein was eluted with elution buffer containing a high concentration of imidazole, according to the manufacturer's instructions.
4
Recombinant ZIKV NS1 Protein Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ZIKV NS1 genes were amplified from the cDNA of infected cells and cloned into a pET-28a (+) expression vector (69864, Millipore). The cloning primers are presented in Extended Data Table 1 . Recombinant NS1 proteins were expressed in the Escherichia coli BL21 DE3 strain in an insoluble form in inclusion bodies. The proteins were then solubilized by 8 M urea and purified with a TALON Purification Kit (635515, Clontech). Murine antisera were produced via the inoculation of recombinant ZIKV NS1 with 3 boosts. Polyclonal antibodies were purified from the immunized antisera using Protein A/G agarose (20423, Thermo). The anti-V5-HRP antibody for tag detection were purchased from Invitrogen (R96125, Thermo).
5
Degradation Kinetics of Floccoderma by P. salicylatoxidans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As previously reported [33] , our laboratory isolated and preserved P. salicylatoxidans CGMCC 1.17248. The protein expression was achieved using Novagen Rosetta (DE3) pLysS cells and a pET28a(+) expression vector from Merck (Kenilworth, New Jersey, USA). E. coli Rosetta strains that overexpress P. salicylatoxidans CGMCC 1.17248 NHase genes (anhA or anhB) had already been constructed by our laboratory [33] .
2.3. Kinetics of FLO Degradation by P. salicylatoxidans CGMCC 1.17248 Resting Cells P. salicylatoxidans CGMCC 1.17248 resting cells were tested for their capacity to degrade FLO. The bacterium was inoculated into 100 mL flasks containing 30 mL of LB medium and incubated for 36 h at 30 • C with shaking at 220 rpm. Following incubation, 3 mL of this culture was inoculated into a 500 mL flask containing 150 mL of LB medium, and a final concentration of 0.1 mmol/L CoCl 2 was added. The culture was incubated for 24 h in the same conditions. Centrifugation at 8000× g for 8 min, followed by two washes with sterilized 50 mmol/L phosphate-buffered saline (PBS, pH 7.5), was used to harvest bacterial cells. After harvesting cells, cell pellets were resuspended in the same buffer containing 0.99 mmol/L FLO, and the optical density of 600 nm was measured and adjusted to 5. HPLC was used to measure residual FLO and its metabolites after samples were collected at the required time intervals.
2.3. Kinetics of FLO Degradation by P. salicylatoxidans CGMCC 1.17248 Resting Cells P. salicylatoxidans CGMCC 1.17248 resting cells were tested for their capacity to degrade FLO. The bacterium was inoculated into 100 mL flasks containing 30 mL of LB medium and incubated for 36 h at 30 • C with shaking at 220 rpm. Following incubation, 3 mL of this culture was inoculated into a 500 mL flask containing 150 mL of LB medium, and a final concentration of 0.1 mmol/L CoCl 2 was added. The culture was incubated for 24 h in the same conditions. Centrifugation at 8000× g for 8 min, followed by two washes with sterilized 50 mmol/L phosphate-buffered saline (PBS, pH 7.5), was used to harvest bacterial cells. After harvesting cells, cell pellets were resuspended in the same buffer containing 0.99 mmol/L FLO, and the optical density of 600 nm was measured and adjusted to 5. HPLC was used to measure residual FLO and its metabolites after samples were collected at the required time intervals.
6
Mycobacterial Strain Engineering and Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The E. coli strain BL21(DE3) (F- ompT hsdSβ(rβ-mβ) dcm gal (DE3) tonA) (Novagen) was used for all protein expression. E. coli K-12 Mach1 T1 cells (ΔrecA1398 endA1 tonA Φ80ΔlacM15 ΔlacX74 hsdR(rk+mk+); Invitrogen) were used for all cloning experiments. M. smegmatis mc2155 and M. tuberculosis H37Rv strains were both used for growth profiles. Experiments involving the virulent H37Rv strains of M. tuberculosis were conducted in a BSL-3 laboratory following institutionally approved protocols. M. tuberculosis H37Rv was also used for the plating efficiency assay to determine recovery of viable bacteria upon VapC-mt11 expression. M. smegmatis mc2155 was used for metabolic labeling. The vapC-mt11 (Rv1561; “mt” refers to M. tuberculosis) gene was cloned using M. tuberculosis H37Rv genomic DNA. The DNA sequences of PCR fragments used for cloning were confirmed by automated DNA sequence analysis. The VapC-mt11 coding region was cloned into the pET28a expression vector (EMD Millipore) and the arabinose inducible pBAD33 plasmid (ATCC) after the addition of 5′ NdeI and 3′ BamHI restriction enzyme sites by PCR. For expression in Mycobacteria, the VapC-mt11 coding region with PCR generated 5′ ClaI and 3′SalI restriction sites was cloned into the corresponding sites in the anhydrotetracycline (ATc) inducible vector pMC1s15 (link).
7
Aspergillus terreus ω-Transaminase Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The gene (gene ID: 115385557) encoding the ω-(R)-transaminase (XP_001209325, Uniprot: Q0C8G1 , [19] (link)) from Aspergillus terreus, which was codon-optimized for E. coli, was ordered from GeneArt (Life Technologies, Carlsbad, CA, USA). The gene was recloned into the pET28a(+) expression vector (Novagen/Merck, Darmstadt, Germany) via the restriction sites NcoI and XhoI to introduce a C-terminal His-Tag. The pET28a-AT-ωTA-CHis construct was confirmed by sequencing (LGC Genomics, Berlin, Germany) and transformed into the expression host E. coli BL21-Gold(DE3) (Stratagene, La Jolla, CA, USA). Mutations were introduced by overlap-extension PCR, and the PCR products were ligated into the vector pMS470Δ8 [31] (link), using NdeI and HindIII restriction sites (primer sequences see Table S1 in File S1 ). The constructs were confirmed by sequencing. E. coli TOP10F’ cells (Life Technologies) were used as expression host.
8
Recombinant Tetanus Neurotoxin Production
9
Cloning and Expression of GFP-Dectin-1 Fusion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human Dectin-1 and Dectin-2 were amplified by PCR using full-length cDNA of human peripheral blood cells as a template. All PCR amplifying fragments, including the Flag-encoding DNA sequence, were inserted into a lentivirus vector, pRV3, between the SalI and BglII sites. To obtain GFP/Dectin-1CRD fusion gene, the method of overlapping PCR was used as described previously (Guan et al., 2007 (link)). Four primers for PCR were designed as follows based on the GFP and the Dectin-1CRD encoding sequence: GFP (sense), 5′-CAGGACATATGGTGAGCAAGGGCGAG-3′; GFP (anti-sense), 5′-GCTGCCACCTCCACCGCTACCGCCGCCTCCCTTGTACAGCTC-3′; Dectin-1CRD (sense), 5′-GGTGGAGGTGGCAGCTTTTGGCGACACAAT-3′; and Dectin-1CRD (anti-sense), 5′-ACTGAGGGATCCCAGTTCCTTCTCA-3′. The GFP (anti-sense) and Dectin-1CRD (sense) primers contained a (Gly4Ser)2 linker encoding sequence (boxed). The fusion PCR product was digested by NdeI and BamHI and was inserted in frame into pET28a expression vector (EMD Millipore).
10
Mutagenesis to Remove NdeI Site in hGNA1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to remove a second NdeI restriction site, a silent mutation, H77H, was introduced into human GNA-1 in the FLAG-HA-hGNA1-pcDNA3.1 plasmid by site-directed mutagenesis48 (link) (primers: hGNA1-H77H_for cttttgagcaCatgaagaaatctgggg, hGNA1-H77H_rev cttcatGtgctcaaaagatttcataaattgttc). Subsequently, GNA-1 was cloned into the pET28a expression vector (Merck Millipore) using NdeI and HindIII restriction sites (primers: hGNA1_NdeI_FOR gagCATATGatgaaacctgatgaaactcctatgtttgaccc, hGNA1_HindIII_REV gagAAGCTTtcactttagaaacctccgacacatgtag).
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!