QIAexpress Type IV Kit

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Publication protocol

NPro Antibody Generation: A region of cDNA encoding the first 130 amino acids of zebrafish N-cadherin (accession number AF418565) was amplified by PCR and then subcloned into the PQE30 expression vector supplied by the Qiaexpress Type IV kit (Qiagen). Subcloned vector DNA was then used to transform M15 (pREP4) competent cells also supplied by the Qiaexpress Type IV kit. Bacterial colonies were screened for positive expression of the NPro-His tagged peptide using an anti-His antibody. High-yield cultures were then generated from this clone and protein expression induced. His-tagged NPro was purified by passing protein purified from the cultures through Ni-NT agarose columns (Qiagen). Purified NPro peptide was then sent to Covance (Denver, Pa) for polyclonal antibody production. Sera were sequentially tested for reactivity as they arrived by western blot analysis using adult zebrafish brain lysates. The most highly reactive sera were used in subsequent studies and for immunohistochemical analysis.



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