Nucleobond xtra midi plus plasmid dna kit

The nucleic acid sequences of ferret, human and mouse BCL6, CXCR5 and PD-1 were extracted from Ensembl website^{22 (link)} and Centre for Biotechnology Information (NCBI). Amino acid homology of ferret, human and mouse BCL6, CXCR5 and PD-1 were compared using Geneious.
The ectodomain of ferret CXCR5 and PD-1 were identified using the NGS dataset (Wong et al. manuscript submitted). The gene sequence of ectodomains of ferret CXCR5 and PD-1 were codon-optimized and synthesized (GeneArt) and cloned into mammalian expression vector containing human IgG1 Fc tag. Plasmids were extracted using NucleoBond Xtra Midi Plus plasmid DNA kit (MACHEREY-NAGEL). Recombinant ferret CXCR5 and PD-1 proteins were expressed by transient transfection of Expi293 (Thermo) suspension cultures with 2.7 μl ExpiFectamine (Thermo) and 1ug DNA/ml cell culture. At day 5 post-transfection, proteins in culture supernatant were purified by protein A agarose affinity chromatography and gel filtration.

The gene of heavy or kappa chain variable domain was synthesized (GeneArt) and cloned into mammalian expression vector containing mouse IgG1 or kappa chain constant domain. Heavy and kappa chain plasmids were extracted using NucleoBond Xtra Midi Plus plasmid DNA kit (MACHEREY-NAGEL). Antibodies were expressed by transient transfection of Expi293 (Thermo) suspension cultures with 2.7 μl ExpiFectamine (Thermo) and 1ug DNA (heavy: kappa = 1:1)/ml cell culture. At day 5 post-transfection, antibodies in culture supernatant were purified by protein G agarose affinity chromatography. For flow cytometric application, antibodies were conjugated to biotin or PE using biotin or PE conjugation kit (Abcam).