cDNA encoding Brme1 (a.a. 1–200) and Brca2 (a.a. 2730–3029) were cloned into the pET28c+ vector (Millipore). The HIS-tagged recombinant proteins were expressed in BL21 (DE3) cells, solubilized in a denaturing buffer (6 mm HCl-guanidine and 30 mm Tris-HCl (pH 7.5)), and purified with Ni-NTA resin (Qiagen). The recombinant proteins were dialyzed in phosphate-buffered saline (PBS) and used to immunize the animals. The polyclonal antibodies were affinity purified on antigen-coupled Sepharose beads (GE Healthcare).
Pet28c vector
Manufactured by Merck Group
The PET28c+ vector is a laboratory tool used for the expression and purification of recombinant proteins. It provides a standard cloning site for inserting the gene of interest and a His-tag sequence for affinity purification of the expressed protein. The vector is designed to work with Escherichia coli (E. coli) as the host organism.
Automatically generated - may contain errors
Lab products found in correlation
Antigen coupled sepharose beads, by GE Healthcare (4 mentions)
Genelute bacterial genomic dna kit, by Merck Group (3 mentions)
Ni nta resin, by Qiagen (2 mentions)
E coli one shot bl21 star de3, by Thermo Fisher Scientific (2 mentions)
Glutathione agarose, by Thermo Fisher Scientific (1 mentions)
Pgex 6p 1 vector, by Addgene (1 mentions)
E coli one shot bl21 de3, by Thermo Fisher Scientific (1 mentions)
7 protocols using pet28c vector
1
Recombinant BRme1 and BRCA2 Protein Purification
2
Recombinant Protein Expression and Antibody Generation
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cDNAs encoding the C-terminus of dtn-1 (a.a. 441–837), the C-terminus of dtn-2 (a.a. 434–818), and full-length pot-2 were cloned into the pET28c+ vector (Millipore). cDNA encoding the C-terminus of pot-1 (a.a. 100–300) was cloned into the pGEX-6P-1 vector (Addgene). The HIS- or GST-tagged recombinant proteins were expressed in BL21 (DE3) cells, solubilized in extraction buffer (600 mM NaCl and 50 mM Tris-HCl (pH 7.5)), and purified with Ni-nitrilotriacetic acid (QIAGEN) for the HIS tag or with glutathione agarose (Thermo Fisher Scientific) for the GST tag. The recombinant proteins were dialyzed into PBS and used to immunize the animals. The polyclonal antibodies were affinity purified on antigen-coupled Sepharose beads (GE Healthcare).
3
Recombinant Protein Expression and Antibody Production
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cDNAs encoding full-length Dmc1, Sycp3, and the N-terminus of Meilb2 (a.a. 1–200) were cloned into the pET28c+ vector (Millipore). The HIS-tagged recombinant proteins were expressed in BL21 (DE3) cells, solubilized in a denaturing buffer (6 M HCl-guanidine and 30 mM Tris-HCl (pH 7.5)), and purified with Ni-nitrilotriacetic acid (QIAGEN). The recombinant proteins were dialyzed in phosphate-buffered saline (PBS) and used to immunize the animals. The polyclonal antibody against MEILB2 was affinity purified on antigen-coupled Sepharose beads (GE Healthcare).
4
Recombinant Protein Expression and Antibody Generation
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cDNAs encoding Dynlrb2 (full length), Ndc80 (full length), Dycn1li2 (amino acids 246–492), and Cenp-E (amino acids 2381–2471) were cloned into the pET28c+ vector (Millipore). The HIS-tagged recombinant proteins were expressed in BL21 (DE3) cells, solubilized in a buffer of 600 mM NaCl, 30 mM imidazole, 20 mM Tris–HCl (pH 7.5), and 0.1% Triton X-100, and purified with Ni-NTA resin (Qiagen). The recombinant proteins were dialyzed in PBS and used to immunize the animals. The polyclonal antibodies were affinity purified on antigen-coupled Sepharose beads (GE Healthcare).
5
Cloning and Mutagenesis of E. coli NudC and NudE
6
Cloning and Expression of E. coli Nud Proteins
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The genes encoding for nudA, nudB, nudC [3 (link)], nudD, nudE, nudF, nudI, nudJ and nudL were PCR-amplified from genomic DNA of E. coli K-12 (isolated via GenElute Bacterial Genomic DNA Kit, Merck KGaA, Darmstadt, Germany). The DNA sequence encoding for the hNUDT5 gene was ordered from IDT and also amplified by PCR as well. pET28a-hDcp2 was ordered from Addgene (72214) (Addgene Europe, Teddington, UK). Restriction sites were introduced during amplification and resulting sequences are listed in Supplementary Table S3 . The resulting PCR products were digested with XbaI/XhoI and NcoI, and cloned into pET-28c vector (Merck Millipore). After Sanger sequencing, the confirmed plasmids were transformed into E. coli One Shot BL21 (DE3) (Thermo Fisher Scientific, Waltham, MA, USA).
7
Cloning and Mutagenesis of E. coli NudC and NudE
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The nudC and nudE gene were PCR-amplified from genomic DNA of E. coli K-12 (isolated via GenElute™ Bacterial Genomic DNA Kit, Sigma-Aldrich). XbaI/NotI (nudC) or XhoI/NcoI (nudE) restriction sites were introduced during amplification using the primers listed in Supplementary Table 2 . The resulting PCR products were digested with XbaI/NotI or XhoI/NcoI and cloned into pET-28c vector (Merck Millipore). After Sanger sequencing, the resulting plasmids were transformed into E. coli One Shot BL21 Star (DE3) (Life Technologies). The NudC mutants were generated by site-directed mutagenesis as described4 (link).
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