Evolutionarily conserved midbody remodeling precedes ring canal formation during gametogenesis. Price et al.
Validation of Hydra KIF23 polyclonal antibody. Western blot from Figure S5.
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A fragment of Hydra KIF23 (Uniprot T2MND8, GenBank accession number XM_012700539.1) from amino acids 208-686 was cloned into the pET30a (+) expression vector. Recombinant protein was expressed in BL21 Star DE3 cells. Purified proteins were used to raise antisera in rabbits (Genscript) from which antibody was then affinity purified. Antibody was verified via Western blot against purified protein that was used as immunogen as well as Hydra whole cell lysate. 50 ng purified protein, 3 Hydra equivalents, and 10 Drosophila testis equivalents were run on a 4-15% gradient SDS-PAGE gel (BioRad) and transferred at 300 mAmps for 42 minutes to a nitrocellulose membrane (GE Healthcare). The blot was probed with 1:5000 anti-KIF23 antibody and two replicates were performed.