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Pet28a vector

Manufactured by Merck Group
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Sourced in Germany, United States

The PET28a vector is a protein expression vector used for the production of recombinant proteins in Escherichia coli. It contains a T7 promoter and a kanamycin resistance gene, allowing for selection of transformed bacterial cells.

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Lab products found in correlation

Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions) Probond puri cation system, by Thermo Fisher Scientific (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Git medium, by Fujifilm (2 mentions) Talon resin, by Takara Bio (2 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions) Bl21 codonplus de3 rilp competent cells, by Agilent Technologies (2 mentions) Pet21a, by Merck Group (2 mentions) Superdex 75 prep grade columns, by GE Healthcare (2 mentions) Bl21 star de3 cells, by Thermo Fisher Scientific (2 mentions) Pcr random mutagenesis kit, by Takara Bio (2 mentions) Escherichia coli hms174 de3, by Merck Group (2 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions) Pib vector, by Thermo Fisher Scientific (1 mentions) Nebuilder hifi dna assembly master mix, by New England Biolabs (1 mentions) Px601, by Addgene (1 mentions) Pacyc184, by New England Biolabs (1 mentions) Lentiguide puro, by Addgene (1 mentions) Pcaggs, by Addgene (1 mentions) Ni nta, by Qiagen (1 mentions)

Close spelling variants of this product

PET28a (83 citations) PET28a expression vector (14 citations) Novagen pET28a(+) vector (6 citations) PET28a empty vector (2 citations)

48 protocols using pet28a vector

1

Construction of PH(1-110)GFP Fusion Protein

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The construction of the fusion protein PH(1-110)GFP (Figure 1A) was designed and previously published by our laboratory (23 (link)). Briefly, the expression vector pFastbacTM1 of the Bac-to-Bac® baculovirus expression system (Thermo Fisher, USA, cat. no. 10359-016) was used for cloning. The 1-110 aa N-terminal coding sequence of polyhedrin (PH(1-110)) was cloned under the polyhedrin (POLH) promoter. The green fluorescent protein (GFP) sequence (GenBank: AAB08058.1) was ligated at the 3’ end of the PH(1-110) sequence. For amplification and titration of the viruses, the manufacturer’s recommendations were followed.
The GFP coding sequence was cloned in open reading frame into the pET-28a(+) vector (Sigma-Aldrich, USA cat. no. 70777) for expression in a bacterial system. The construct added an N-terminal His-tagged and the Kanamycin resistance gene. The same procedure was performed to clone the PH(1-110) sequence into the pET-28a(+) vector (Sigma-Aldrich, USA cat. no. 70777).
Cruz-Resendiz A., Acero G., Sampieri A., Gevorkian G., Salvador C., Escobar L., Rosendo-Pineda M.J., Medeiros M, & Vaca L. (2022). An ambient-temperature stable nanoparticle-based vaccine for nasal application that confers long-lasting immunogenicity to carried antigens. Frontiers in Immunology, 13, 1057499.
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2

Purification of MmMARF1 Protein

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The coding sequence of MmMARF1 (residues 158–320, 158–407, 158–690, 158–1381, and 687–1381) were cloned into the modified pET28a vector with an N-terminal His6-SUMO tag (residues 158-320, 158-407, and 158-690; Merck), the pfastBacHTB vector (residues 158-1381; Thermo Fisher Scientific, Inc.), and the pET28a vector with a C-terminal His6 tag (residues 687-1381; Merck), respectively, and the recombinant protein was then produced and purified as described in SI Appendix, Materials and Methods.
Yao Q., Cao G., Li M., Wu B., Zhang X., Zhang T., Guo J., Yin H., Shi L., Chen J., Yu X., Zheng L., Ma J, & Su Y.Q. (2018). Ribonuclease activity of MARF1 controls oocyte RNA homeostasis and genome integrity in mice. Proceedings of the National Academy of Sciences of the United States of America, 115(44), 11250-11255.
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3

Plasmid construction for Par-1 and LacZ expression

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Plasmids pIB-3×Myc and pIB-3×Flag were generated using a pIB vector (Thermo Fisher Scientific). Vectors to express Flag-Par-1 were generated by inserting Par-1 cDNAs into pIB-3×Flag using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). A DNA fragment encoding β-galactosidase was PCR-amplified from pMT/V5-His/lacZ (Thermo Fisher Scientific) and cloned into the PCR amplified pIB-3×Flag vector. Then, the expression vector of F-LacZ was generated using NEBuilder HiFi DNA Assembly Master Mix. Vectors for expressing the Papi-Flag mutants (S157A, S547A, S565A, ΔMLS, S547D, S547E, 1-222, and 1-480) were generated by inverse PCR, using RNAi-resistant Papi-Flag14 (link) as the template. A DNA fragment encoding Papi-Flag WT and 1-480 were PCR-amplified from pIB-Papi-3×Flag and pIB-Papi-1-480-3×Flag respectively and cloned into the PCR amplified pET-28a(+) vector (Sigma) Then, the vectors of Papi-F WT and 1-480 for TnT® was generated using NEBuilder HiFi DNA Assembly Master Mix. The PCR primers used are summarized in Supplementary Table S1.
Yamada H., Nishida K.M., Iwasaki Y.W., Isota Y., Negishi L, & Siomi M.C. (2022). Siwi cooperates with Par-1 kinase to resolve the autoinhibitory effect of Papi for Siwi-piRISC biogenesis. Nature Communications, 13, 1518.
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4

Type I-F CRISPR System Plasmid Construction

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Type I–F Cascade (from Pseudomonas aeruginosa) E. coli expression plasmids were obtained from Addgene (pCsy_complex, 89232). Type I–Fv (from Shewanella putrefaciens) Cas7fv, Cas5fv, Cas6fv cassettes were cloned into the pET28a vector (Sigma-Aldrich, 69864-3CN) as a polycistronic operon and include an N-terminal His-tagged Cas7fv fusion (pET28-type I–Fv). The crRNA sequence was cloned into pACYC184 (NEB, X06403) for bacterial expression. Condon-optimized Cas subunits were sub-cloned into px601 (Addgene, #61591) (replacing the SaCas9 gene) for transfection into mammalian cells. A site for spacer cloning flanked by two Csy4 direct repeats (DR) or Cas6f direct repeats was ligated into lentiGuide-Puro (addgene #52963) between BsmBI and EcoRI restriction sites to generate pLenti-crRNA-IF or pLenti-crRNA-IFv vectors. Oligos containing spacer sequences were annealed and ligated into pLenti-crRNA-IF or pLenti-crRNA-IFv for crRNA expression in mammalian cells. For spacer mutant crRNA cloning, oligos with various of mutant spacer were annealed and ligated into pLenti-crRNA-IF. Sequences are listed in Supplementary Data 1–4. Sequences of plasmids for expression of PaeCascade-VPR, including pCsy1-Csy2, pCsy3-VPR-Csy4, and pCsy-crRNA-EV, are listed in Supplementary Data 5.
Chen Y., Liu J., Zhi S., Zheng Q., Ma W., Huang J., Liu Y., Liu D., Liang P, & Songyang Z. (2020). Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells. Nature Communications, 11, 3136.
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5

Recombinant BbiTRAP-1 Protein Production

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For ease of expression, predicted transmembrane regions of high hydrophobicity of BbiTRAP-1 were identi ed using the TMHMM Server v.2.0 (www.cbs.dtu.dk/services/TMHMM) and removed from the nal sequence. Therefore, a truncated DNA fragment of 1875bp coding for 625 amino acids (corresponding to amino acids 320 to 944) was commercially synthesized (GenScript, USA) and cloned between the EcoRI and BamHI sites in the pET-28a vector (Sigma-Aldrich, Steinheim, Germany) along with an in-frame 6X Histidine tag for further protein puri cation. The plasmid map can be accessed in https://www.addgene.org/vector-database/2565/.
The BbiTRAP-1-pET28a construct was used to transform chemically competent E. coli Rosetta (DE3) BL21 strain (Invitrogen, Carlsbad, CA, USA). Maximal expression of the protein was obtained after induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 20 °C overnight. Protein puri cation was performed according to the manufacturer's instructions (ProBond™ Puri cation System, Life Technologies, Carlsbad, CA, USA) for puri cation of poly Histidine-containing recombinant proteins. The puri ed BbiTRAP-1 protein was analyzed by SDS-PAGE and the protein identity was con rmed by Western blot using an anti-His tag commercial antibody (Abcam, Cambridge, USA). The protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scienti c, USA).
Montenegro V.N., Paoletta M.S., Ortiz J.J., Suárez C.E., & Wilkowsky S.E. (2020). Identification and characterization of a Babesia bigemina thrombospondin-related superfamily member, TRAP-1: a novel antigen containing neutralizing epitopes involved in merozoite invasion.
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6

Recombinant BbiTRAP-1 Protein Purification

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For ease of expression, predicted transmembrane regions of high hydrophobicity of BbiTRAP-1 were identi ed using the TMHMM Server v.2.0 (www.cbs.dtu.dk/services/TMHMM) and removed from the nal sequence. Therefore, a truncated DNA fragment coding for of 625 amino acids (corresponding to amino acids 320 to 944) was commercially synthesized (GenScript, USA) and cloned in the pET-28a vector (Sigma-Aldrich, Steinheim, Germany) along with an in-frame 6X Histidine tag for further protein puri cation.
The BbiTRAP-1-pET28a construct was used to transform chemically competent E. coli Rosetta (DE3) BL21 strain (Invitrogen, Carlsbad, CA, USA). Maximal expression of the protein was obtained after induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 20°C overnight. Protein puri cation was performed according to the manufacturer's instructions (ProBond™ Puri cation System, Life Technologies, Carlsbad, CA, USA) for puri cation of poly Histidine-containing recombinant proteins. The puri ed BbiTRAP-1 protein was analyzed by SDS-PAGE and the protein identity was con rmed by Western blot using an anti-His tag commercial antibody (Abcam, Cambridge, USA). The protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scienti c, USA).
Montenegro V.N., Paoletta M.S., Ortiz J.J., Suárez C.E., & Wilkowsky S.E. (2020). Identification and characterization of a Babesia bigemina thrombospondin-related superfamily member, TRAP-1: a novel antigen containing neutralizing epitopes involved in merozoite invasion.
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7

Generation of Anti-Celf3 Monoclonal Antibody

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To produce the His-tagged recombinant proteins His-Celf3, the corresponding cDNA was subcloned into the BamHI/SalI sites of the pET-28a vector (Millipore). The recombinant protein was produced in E. coli BL21 (DE3) and purified with TALON resin (Clontech). For immunization, His-Celf3 (10–50 μg) was mixed with the adjuvant TiterMax Gold (TiterMax) to produce antigen-adjuvant emulsions (100 μL/mouse) and injected intraperitoneally into four BALB/c female mice (8 weeks old at the first injection) every 2 weeks. The lymphocytes from the immunized mice were fused with myeloma P3U1 cells in a ratio of 3:1 to 5:1 by mixing in 50% polyethylene glycol (Roche). The fused cells were dispersed into 80 mL GIT medium (Wako, Japan) supplemented with 1 ng/mL IL-6 (PeproTech) and 1 × HAT (Kohjin-Bio). The cells were seeded into four 96-well plates at 0.2 mL/well and grown for 10 days at 37 °C. The first screening was carried out by enzyme-linked immunosorbent assay (ELISA) with 50 ng/well of His-Celf3 (37 positive clones were obtained), and the clones were subsequently screened with Western blot, immunofluorescence and immunoprecipitation analyses. As a result, clone 1E7 was selected as the anti-Celf3 monoclonal antibody for use in this study. Antiserum against Celf3 was prepared at the fusion step.
Ishizuka A., Hasegawa Y., Ishida K., Yanaka K, & Nakagawa S. (2014). Formation of nuclear bodies by the lncRNA Gomafu-associating proteins Celf3 and SF1. Genes to Cells, 19(9), 704-721.
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8

Production and Screening of Anti-Tanmp Antibodies

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For production of the His-tagged recombinant protein His-Tanmp, the corresponding cDNA was subcloned into the pET-28a vector (Millipore). The recombinant protein was produced in Escherichia coli BL21 (DE3) and purified with TALON resin (Clontech). For immunization, 10 µg of His-Tanmp was mixed with the adjuvant TiterMax Gold (TiterMax) to produce antigen-adjuvant emulsions and injected intraperitoneally into four BALB/c female mice every two weeks. The lymphocytes from the immunized mice were fused with myeloma P3U1 cells at a ratio of 3:1 by mixing in 50% polyethylene glycol (Roche). The fused cells were dispersed in 80 ml of GIT medium (Wako, Japan) supplemented with 1 ng/ml IL-6 (PeproTech) and 1×HAT (Kohjin-Bio). The cells were seeded in four 96-well plates at 0.2 ml/well and grown for 10 days at 37°C. The first screening was performed by enzyme-linked immunosorbent assay (ELISA) with 50 ng/well His-Tanmp and subsequently screened with Western blots and immunofluorescence.
Inanami O., Tanahashi M., Yokoi S., Kaneko M., Tokuhara T., Yanaka K., Nakagawa S., & Maita H. (2021). Identification of the cell-type-specific ER membrane protein Tanmp expressed in hypothalamic tanycytes and subsets of neurons.
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9

Recombinant Ferritin Protein Purification

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Full-length ferritin heavy chain and light chain cDNA was cloned into pET-21a (+) and pET-28a (+) vectors (Millipore), respectively. pET-21 ferritin heavy chain construct and pET-28 ferritin light chain construct were transformed into BL21-CodonPlus (DE3)-RILP Competent Cells (Agilent) sequentially. For protein expression, positive transformants were grown in LB medium with carbenicillin and kanamycin overnight. The saturated culture was diluted in the following day to obtain an exponential-phase culture, to which 1.0 mM IPTG was added to induce ferritin expression for 2.5 hours at 37°C. Cells were pelleted and sonicated, and supernatant was heated at 70°C for 15 mins. Ferritin was isolated from the supernatant by centrifugation. Ferritin was purified by fast protein liquid chromatography (FPLC) using a HiPrep 26/60 Sephacryl S-300 HR gel filtration column (Sigms) filled with 50 mM Tris pH=7.5, 50 mM NaCl buffer followed by a HiTrap Q HP ion exchange column filled with 50 mM Tris pH 7.5 buffer with a 50–300 mM NaCl gradient. Purified protein was dialyzed to the buffer containing 50 mM Tris pH 7.5 and 50 mM NaCl for storage.
Cheung P., Schaffert S., Chang S.E., Dvorak M., Donato M., Macaubas C., Foecke M.H., Li T.M., Zhang L., Coan J.P., Schulert G.S., Grom A.A., Henderson L.A., Nigrovic P.A., Elias J.E., Gozani O., Mellins E.D., Khatri P., Utz P.J, & Kuo A.J. (2021). REPRESSION OF CTSG, ELANE, AND PRTN3-MEDIATED HISTONE H3 PROTEOLYTIC CLEAVAGE PROMOTES MONOCYTE-TO-MACROPHAGE DIFFERENTIATION. Nature immunology, 22(6), 711-722.
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10

Recombinant Ferritin Protein Purification

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Full-length ferritin heavy chain and light chain cDNA was cloned into pET-21a (+) and pET-28a (+) vectors (Millipore), respectively. pET-21 ferritin heavy chain construct and pET-28 ferritin light chain construct were transformed into BL21-CodonPlus (DE3)-RILP Competent Cells (Agilent) sequentially. For protein expression, positive transformants were grown in LB medium with carbenicillin and kanamycin overnight. The saturated culture was diluted in the following day to obtain an exponential-phase culture, to which 1.0 mM IPTG was added to induce ferritin expression for 2.5 hours at 37°C. Cells were pelleted and sonicated, and supernatant was heated at 70°C for 15 mins. Ferritin was isolated from the supernatant by centrifugation. Ferritin was purified by fast protein liquid chromatography (FPLC) using a HiPrep 26/60 Sephacryl S-300 HR gel filtration column (Sigms) filled with 50 mM Tris pH=7.5, 50 mM NaCl buffer followed by a HiTrap Q HP ion exchange column filled with 50 mM Tris pH 7.5 buffer with a 50–300 mM NaCl gradient. Purified protein was dialyzed to the buffer containing 50 mM Tris pH 7.5 and 50 mM NaCl for storage.
Cheung P., Schaffert S., Chang S.E., Dvorak M., Donato M., Macaubas C., Foecke M.H., Li T.M., Zhang L., Coan J.P., Schulert G.S., Grom A.A., Henderson L.A., Nigrovic P.A., Elias J.E., Gozani O., Mellins E.D., Khatri P., Utz P.J, & Kuo A.J. (2021). REPRESSION OF CTSG, ELANE, AND PRTN3-MEDIATED HISTONE H3 PROTEOLYTIC CLEAVAGE PROMOTES MONOCYTE-TO-MACROPHAGE DIFFERENTIATION. Nature immunology, 22(6), 711-722.
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