NES (Nestin)
Antibody Concentrations: 300 µg/ml (based on Bradford assay readings using bovine serum albumin as a standard).
Volume: Regular vials contain 1000 µl; Sampler vials contain 200 µl.
Buffer: Phosphate-buffered (10 mM) isotonic (0.9%, w/v) saline (PBS, pH 7.2) with sodium azide (0.02%, w/v) added as a preservative.
Quality Control: Immunohistochemistry (at a concentration of 3 µg/ml) using fluorescein-labeled goat anti-chicken IgY (1:500 dilution, Aves Labs Cat. #F-1004) as the secondary reagent.
Recommended Dilutions: 1:10000-1:20000 for immunohistochemistry and immunocytochemistry using 2% paraformaldehyde-fixed tissues or cells. 1:20000-1:50000 for westerns.
Chickens were immunized with three synthetic peptide / keyhole limpet hemocyanin (KLH) conjugates. These synthetic peptides corresponded to different regions of the mouse nestin gene product (NP_057910). [Note that the sequences of the mouse and human nestin genes are divergent, and it is unlikely that antibodies raised against the mouse sequence would recognize human nestin.] After repeated injections into the hens, immune eggs were collected, and the IgY fractions were purified from the yolks. These IgY fractions were then affinity-purified using a peptide column, and the concentrations of the eluates adjusted to 300 µg/ml. Finally, equal volumes of each of the three affinity-purified anti-peptide antibodies were mixed, and the preparation was filter-sterilized.