Human LMPTP-A was purified as previously reported (14 (link)). A codon-optimized open reading frame for bacterial expression of full-length WT GSS as a thrombin cleavable 6xHistidine N-terminal fusion in pET28a vector was purchased from Genscript. Escherichia coli BL21 (DE3) cell cultures were grown at 37°C in Luria-Bertani broth and induced for 16 to 20 hours at 18°C by the addition of 0.25 mM isopropyl-β-d -thiogalactopyranoside. An amount of target protein sufficient for the isolation of several milligrams of protein was present in the soluble fraction of the lysate from one liter of culture. Briefly, the cleared lysate in 20 mM tris-HCl (pH 8.0), 300 mM NaCl was applied to a nickel-nitrilotriacetic acid affinity column (QIAGEN) by gravity flow and eluted with successive buffers containing increasing amount of imidazole, followed by a polishing step by Superdex 200 (Cytiva) size exclusion chromatography (SEC). For crystallization, the 6xHis tag was removed with thrombin before the SEC step. The final purity was assessed to be >95% by SDS-PAGE, and the protein was concentrated to 8 to 10 mg/ml and stored at −80°C. All mutants were purchased from GenScript or obtained by standard site-directed mutagenesis techniques and confirmed by sequencing. All primers used were synthesized by Integrated DNA Technologies.
Nickel nitrilotriacetic acid affinity column
Manufactured by Qiagen
The Nickel-nitrilotriacetic acid affinity column is a laboratory tool used for the purification and isolation of recombinant proteins containing a histidine-tag. The column utilizes the high affinity between the histidine-tag and the immobilized nickel ions, allowing targeted proteins to be selectively captured and separated from other components in a sample.
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3 protocols using nickel nitrilotriacetic acid affinity column
1
Bacterial Expression and Purification of GSS
2
Recombinant Flagellar Protein Expression
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Various insert DNAs were prepared by amplifying the following DNA fragments: the complete ORFs for DegQ, FlaJ, and FlaA to D; several flaB-derived sequences truncated in various N- and/or C-terminal domains (i.e., ΔN0, ΔN1, ΔC0, ΔC1, ΔN0C0, ΔN1C0, and ΔN1C1); and several flaC-derived sequences having a single or multiple point mutations (i.e., M65V, G365S, M380L, and M65V/M157V/M159L/M380L). In cases of the PCR for the flaC-derived sequences, the overlap extension method56 (link) was applied to accomplish the site-directed mutagenesis events during amplification. The lists of the primer sets for PCR are summarized Supplementary Table 2 . Resultant PCR products were cloned into pQE30 and transformed into E. coli JM109. All the mutagenized nucleotide sequences constructed in this study were confirmed by DNA sequencing. The recombinant proteins overexpressed in the presence of 1.0 mM IPTG were purified using a nickel-nitrilotriacetic acid affinity column (Qiagen) and utilized for the in vitro proteolysis assay. Purified recombinant proteins of rFlaJ and rDegQ were used to raise the polyclonal antibodies by treating 50 μg proteins to 6-week-old female Sprague-Dawley rats. The animals received humane care in accordance with our institutional guidelines and the legal requirements (Sogang Univ. IACUCSGU2019_01).
3
Molecular Characterization of Vibrio Flagellins
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To amplify DNA fragments containing the complete ORFs of the flaA, -B, -C, -E, and -F genes of V. vulnificus , the flaC, -E, and -F genes of V. parahaemolyticus , and the flaA and -E genes of V. cholerae , specific primer sets were used (Table S2 ). Each PCR product was cloned into pQE30 and transformed into E. coli JM109. Overexpressed recombinant proteins in the presence of 1.0 mM IPTG were purified using a nickel-nitrilotriacetic acid affinity column, as per the manufacturer’s instructions (Qiagen). Purified recombinant proteins were verified by SDS-PAGE prior to addition to the biofilm formation and EPS interaction assays. Purified recombinant proteins of V. vulnificus were also used to raise the polyclonal antibodies by treating 6-week-old female Sprague-Dawley rats with 50-μg quantities of flagellins or FHPs (the animals received humane care in accordance with our institutional guidelines and the legal requirements [IACUCSGU2019_01]).
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