Resource Report

URL: http://antibodyregistry.org/AB_2877176

Proper Citation: (Maurice Elphick - Queen Mary University of London, UK Cat# ArCCK/SK1, RRID:AB_2877176)

Target Antigen: ArCCK/SK1 sulphated tyrosine

Host Organism: rabbit

Clonality: polyclonal

Comments: "To generate antibodies to the starfish (Asterias rubens) neuropeptide ArCCK/SK1 (previously known as ArCCK1; Semmens et al. 2016; https://doi.org/10.1098/rsob.150224), an N-terminally truncated peptide analog of ArCCK/SK1 with the addition of a reactive N-terminal lysine residue was synthesized as an antigen (KY(SO3H)GHGLFW-NH2, Peptide Protein Research Ltd, Fareham, UK). This peptide was conjugated to porcine thyroglobulin (Sigma-Aldrich, Gillingham, UK) as a carrier protein using 5% glutaraldehyde in phosphate buffer (0.1 M; pH 7.2) and the conjugate was used for immunisation of a rabbit (70-day protocol; Charles River Biologics, Romans, France). The antigen was emulsified in Freund’s complete adjuvant for primary immunisations (~100 nmol antigen peptide) and in Freund’s incomplete adjuvant for three booster immunisations (~50 nmol antigen peptide). The presence of antibodies to the antigen peptide in post-immunisation serum samples was assessed using an enzyme-linked immunosorbent assay (ELISA), in comparison with pre-immune serum. Antibodies to the antigen peptide were purified from the final bleed antiserum by affinity-purification using the AminoLink Plus Immobilization Kit (ThermoFisher Scientific, Waltham, MA, USA), with bound antibodies eluted using glycine elution buffer [6.3 ml of 100 mM glycine (VWR Chemicals, Leicestershire, UK) and 0.7 ml of Tris (1M, pH = 7.0)] and trimethylamine (TEA) elution buffer [6.3 ml of TEA (Sigma-Aldrich, Gillingham, UK) and 0.7 ml of Tris (1M, pH = 7.0)]. Eluates were dialyzed and sodium azide (0.1%) was added for long-term storage of the affinity-purified antibodies at 4oC."