The genes of candidate marker antigens were amplified by polymerase chain reaction (PCR) using the plasmid DNA containing the candidate marker antigen gene as templates. Six histidine residues (His) were added at the N- and C-terminus of genes during PCR amplification. The target genes were cloned into the pFastBac1 vector (Invitrogen) and confirmed by sequencing. The recombinant bacmid was generated using the Bac-to-Bac System (Invitrogen). The recombinant baculovirus was generated by transfecting the recombinant bacmid to Sf9 cells using TransIT-Insect Transfection Reagent (Mirus Bio). The recombinant proteins that were extracted from baculovirus-infected cells were then analyzed by SDS-PAGE and western blotting (WB). Finally, the proteins were purified using a His-tagged protein purification kit (CoWin Biosciences).
His tagged protein purification kit
Manufactured by CoWin Biotech
Sourced in China
The His-tagged protein purification kit is a laboratory equipment designed for the efficient purification of recombinant proteins containing a histidine (His) tag. The kit utilizes the high affinity between the His-tag and nickel-charged resin to capture and isolate the target protein from complex mixtures. The purified protein can then be eluted and collected for further analysis or applications.
Automatically generated - may contain errors
Lab products found in correlation
Toxineraser endotoxin removal kit, by GenScript (2 mentions)
Toxinsensor chromogenic lal endotoxin assay kit, by GenScript (2 mentions)
Bac to bac system, by Thermo Fisher Scientific (1 mentions)
Pfastbac1 vector, by Thermo Fisher Scientific (1 mentions)
Transit insect transfection reagent, by Mirus Bio (1 mentions)
Hindiii, by Takara Bio (1 mentions)
E colibl21 de3 cells, by Takara Bio (1 mentions)
Bamhi, by Takara Bio (1 mentions)
Escherichia coli dh5α competent cells, by Takara Bio (1 mentions)
5 protocols using his tagged protein purification kit
1
Recombinant Protein Production Protocol
2
Recombinant Odorant-Binding Protein Expression
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The sequences of SfurOBP1, SfurOBP2, SfurOBP3 and SfurOBP11 were downloaded from NCBI GenBank (GenBank accession numbers KF732013 , KF660218 , KF732014 and KF732020 , respectively). The SfurOBP genes were amplified by gene-specific primers (Table S1 ) and cloned into the vector pET-30a (+) using BamH I (TaKaRa) and Hind III (TaKaRa) restriction endonucleases. The pET-30a (+) vector allowed the expression of a recombinant product tagged with a His-tag sequence at the N-terminus. The recombinant plasmids were transformed into Escherichia coli DH5α competent cells (TaKaRa). The confirmed plasmids were transformed into E. coli BL21(DE3) cells (TaKaRa). The expression of recombinant proteins was induced with a final concentration of 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Recombinant proteins were purified by His-Tagged Protein Purification Kit (Cowin Biotech Co. Ltd., Beijing, China) and His-tag was removed using recombinant enterokinase (Novagen, Madison, WI, USA), according to the manufacturer’s protocols. Protein expression and purification were monitored by 15% SDS-PAGE. The concentration of highly purified proteins was determined by the standard bicinchoninic acid (BCA) method (Sangon Biotech Co. Ltd., Shanghai, China).
3
RAD51 Acetylation Assay Protocol
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His-tagged RAD51 was purified by His-tagged Protein Purification Kit as the manufacturer's procedure (CoWin Biosciences, China) . In 50 μl reaction buffer containing His-tagged RAD51 5 μg, 8 μg HeLa cell lysate as acetyltransferase donor, 50 μM Tris-HCL (pH = 8.0), 20 μM acetyl-CoA, 0.1 mM EDTA, 1 mM DTT, and 10% Glycerol was constructed and incubated at 37°C for 1 h. Then, the samples were divided into two aliquots for immunoblot, and Coomassie dyed the gel. The proteins in gel bands were excised and then sent to Oebiotech for mass spectrometric analysis.
4
Recombinant Mouse Interleukin-33 Production
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Full-length mouse IL-33 was amplified from mouse spleen cDNA and cloned into a pUC-T vector (Beijing CoWin Biotech, Beijing, China) as previously described [16 (link)]. In brief, the insert was confirmed by direct DNA sequencing. The cDNA sequence, starting with amino acid 112 of the full-length protein, was then amplified from the above plasmid containing the mature IL-33 using specific primer pairs: 5′-GCATGAATTCATGACATTGAGCATCCAAGGAAC-3′ (forward) and 5′-CCGCCTCGAGGATTTTCGAGAGCTTAAACA-3′ (reverse). The resulting amplified fragment was inserted into the expression vector pET21a (+) at the EcoRI and XhoI to yield the construct pET21a-IL-33. This construct was transformed into Escherichia coli strain BL21DE3, and the expression of rIL-33 was induced by isopropyl-β-D-thiogalactoside and purified by using 6×His-Tagged Protein Purification Kit (Beijing CoWin Biotech, Beijing, China), followed by ToxinEraser Endotoxin Removal Kit (GenScript, Nanjing, China) to remove any endotoxin that might have come from the host cells. The purity of rIL-33 was more than 95% tested by SDS-PAGE, and the endotoxin levels were less than 1 Eu/mg of protein using Toxin Sensor Chromogenic LAL Endotoxin Assay Kit (GenScript, Nanjing, China).
5
Recombinant Mouse IL-33 Protein Production
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Full-length mouse IL-33 was amplified from mouse spleen cDNA and cloned into a pUC-T vetor (Beijing CoWin Biotech, Beijing, China). The insert was confirmed by direct DNA sequencing. The cDNA sequence, starting with amino acid 112 of the full-length protein, was then amplified from the above plasmid containing the mature IL-33 using specific primer pairs: 5′-GCATGAATTCATGACATTGAGCATCCAAGGAAC-3′ (forward) and 5′-CCGCCTCGAGGATTTTCGAGAGCTTAAACA-3′ (reverse). The resulting amplified fragment was inserted into the expression vector pET21a (+) at the EcoR I and Xho I to yield the construct pET21a-IL-33. This construct was transformed into Escherichia coli strain BL21DE3, and the expression of rIL-33 was induced by isopropyl-β-d-thiogalactoside and purified by using 6 × His-Tagged Protein Purification Kit (Beijing CoWin Biotech, Beijing, China), followed by ToxinEraser Endotoxin Removal Kit (GenScript, Nanjing, China) to remove any endotoxin that might have come from the host cells. The purity of rIL-33 was more than 95% tested by SDS-PAGE, and the endotoxin levels were less than 1 Eu/mg of protein using ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript, Nanjing, China).
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