Ptrchis vector

Manufactured by Thermo Fisher Scientific

The PTrcHis vector is a plasmid designed for the expression and purification of recombinant proteins in Escherichia coli. It contains a trc promoter, which is a hybrid of the trp and lac promoters, and a 6xHis tag sequence for affinity-based protein purification.

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Lab products found in correlation

Pgex 4t vector, by GE Healthcare (1 mentions) Ni nta column, by Qiagen (1 mentions)

Close spelling variants of this product

PTrcHis TA vector PTrcHis TOPO vector PTrcHis

4 protocols using ptrchis vector

Purification of Arabidopsis NPR1 and TGA3

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Full-length Arabidopsis NPR1 was cloned into the pTrcHis vector (Thermo Fisher Scientific) as a His₁₀–MBP–fusion protein containing a PreScission protease site following MBP and a C-terminal Strep tag (Strep3) with the last Lys residue of the Strep tag mutated to Arg. Transformed BL21(DE3) E. coli cells were grown in Luria–Bertani (LB) medium at 37 °C to an optical density at 600 nm (OD₆₀₀) of 0.5, induced with 0.1 mM IPTG, 0.1 mM ZnSO₄ and 0.2 mM SA at 18 °C overnight. The purification of the E. coli-expressed NPR1 was performed as described above for the insect-cell-expressed NPR1, except that the purification buffer contained 0.2 mM SA.
Arabidopsis TGA3 (Asn87–Thr384) was cloned into the pTrcHis vector (Thermo Fisher Scientific) as a His₁₀–MBP fusion protein containing a PreScission protease site following MBP and a C-terminal tandem Flag–HA tag. Transformed BL21(DE3) E. coli cells were grown in LB medium at 37 °C to an OD₆₀₀ of 0.5 and induced with 0.1 mM IPTG at 18 °C overnight. The purification was performed as described above for NPR1.

Kumar S., Zavaliev R., Wu Q., Zhou Y., Cheng J., Dillard L., Powers J., Withers J., Zhao J., Guan Z., Borgnia M.J., Bartesaghi A., Dong X, & Zhou P. (2022). Structural basis of NPR1 in activating plant immunity. Nature, 605(7910), 561-566.

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NPR1 BTB/BHB Oxidation Assay

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WT NPR1 BTB/BHB (Thr39–Lys262) and the npr1(C156A), npr1(C82A) and npr1(C212V/C216A/C223L) mutants were cloned into the pTrcHis vector (Thermo Fisher Scientific) as His₁₀–MBP fusion proteins with a PreScission protease site following MBP and a C-terminal Strep tag. The proteins were expressed and purified similarly to the NPR1(ΔSBD) as described above. The NPR1 BTB/BHB WT and mutant proteins at a concentration of 25 μM were incubated with 1 mM hydrogen peroxide in a buffer containing 125 mM HEPES and 150 mM NaCl at pH 8.0 at room temperature for 45 min. The oxidized samples were incubated with 50 mM iodoacetamide for 5 min, mixed with 4× SDS loading dye and analysed using SDS–PAGE in the absence of any reducing reagent.

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Purification of Syt-1, Syt-7 C2AB Domains

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C2AB modules from Syt-1 and Syt-7 chimeras (amino acids 96–421) were subcloned into a pGEX4T vector (GE), expressed as GST-fusion proteins, purified from Escherichia coli lysates via GST-sepharose affinity chromatography, and thrombin cleaved in 100 mM KCl, 25 mM HEPES, pH 7.4, 5% glycerol. Syt7 C2AB (amino acids 134–403) was expressed with a His₆ tag (pTrcHis vector, Invitrogen), purified from E. coli lysates via Ni-NTA-sepharose affinity chromatography, eluted with 500 mM imidazole, and dialyzed overnight against 100 mM KCl, 25 mM HEPES, pH 7.4, 5% glycerol. Protein concentrations were determined by SDS–PAGE using BSA standards.

Bendahmane M., Bohannon K.P., Bradberry M.M., Rao T.C., Schmidtke M.W., Abbineni P.S., Chon N.L., Tran S., Lin H., Chapman E.R., Knight J.D, & Anantharam A. (2018). The synaptotagmin C2B domain calcium-binding loops modulate the rate of fusion pore expansion. Molecular Biology of the Cell, 29(7), 834-845.

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TbKINX1B C-terminus Protein Purification

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The C′-terminus of TbKINX1B (aa 821–1342) was expressed in E. coli XL1Blue bacteria, using the pTrcHis vector (Invitrogen). This truncation contained an N-terminal His-tag. The protein was purified under native conditions on NI-NTA columns (Qiagen) and used to inject three BALB/c mice. Mice were inoculated with the purified protein as follows: first injection: 50 μg in PBS mixed with complete Freund’s adjuvant, intraperitoneally (IP). Second and third injections: 25 μg mixed with incomplete Freund’s Adjuvant (IP). Fourth injection, 25 μg in PBS, (IP). Injections were done at intervals of three weeks and the mouse with best serum response by ELISA assays was used for myeloma fusion. Fusion of spleen and myeloma cells (P3X63-Ag8.653), was PEG-induced. Screening of hybridoma culture supernatants was done initially by ELISA assay, and positive clones were then screened by Western blot and immunofluorescence. The resulting antibody is an IgM (kappa light chain). Supernatants were collected and precipitated with 50% ammonium sulphate.

Perdomo D., Berdance E., Lallinger-Kube G., Sahin A., Dacheux D., Landrein N., Cayrel A., Ersfeld K., Bonhivers M., Kohl L, & Robinson D.R. (2022). TbKINX1B: a novel BILBO1 partner and an essential protein in bloodstream form Trypanosoma brucei. Parasite, 29, 14.

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