Ab2231

Goat anti-DDAH-1 primary antibody (1:100; ab2231; Abcam) was preabsorped with 10 times excess (weight/weight) of the peptide used to generate the antibody (ab99047; Abcam) overnight at 4°C. The mixture was then centrifuged for 20 minutes at 10,000 rpm and the top half of the solution was collected and applied to tissue sections. A positive control (antibody alone at the same dilution) and a negative control (lack of primary antibody) were run in parallel.

Urethane-anesthetized rats were killed by decapitation and tissues (spinal dorsal horn, DRG, and hippocampus) were dissected and snap frozen. Tissues were homogenized in RIPA (radioimmunoprecipitation assay) buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS + 0.5% deoxycholic acid + complete protease inhibitor cocktail) using a glass homogenizer. Homogenates were centrifuged at 14,000 rpm for 10 minutes at 4°C, and supernatants containing whole-cell tissue lysates were collected. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Cramlington, United Kingdom). Laemmli loading buffer was added to protein lysates (40 μg) and samples were incubated at 70°C for 30 minutes, before loading onto 8% gels, and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Proteins were transferred to nitrocellulose membranes and probed overnight at 4°C with goat anti-DDAH-1 (1:500; ab2231; Abcam, Cambridge, United Kingdom) or rabbit anti-neuronal β-III Tubulin (1:3000; ab18207; Abcam), which served as a loading control. Membranes were incubated with IRDye-linked donkey anti-goat 680 or donkey anti-rabbit 800CW secondary antibody (1:15,000) for 1 hour at RT. Proteins were revealed using the Odyssey fluorescence detection system (Licor, Cambridge, United Kingdom).