Western blotting of formaldehyde-fixed neuropeptides as small as 400 daltons on gelatin-coated nitrocellulose paper

A method is described for Western blotting of peptides as small as 400 daltons (Da), Peptides were separated by tricine-sodium dodecyl sulfate electrophoresis and electroblotted to gelatin-coated PH79 nitrocellulose paper (0.1 μm). The electroblotted peptides were fixed to the nitrocellulose paper for 5-10 min in 4% paraformaldehyde solution. Using anti-rabbit FMRF-amide (Phe-Met-Arg-Phe-NH₂) as primary antibody, positive immunoreactivity was detected with an amplified alkaline phosphatase assay which was sensitive to at least 0.5 μg FMRFamide/lane. When immunoreactivity was determined with ¹²⁵I-protein A, it was possible to amplify and detect weak signals by increasing the autoradiography time. Therefore, using the ¹²⁵I-protein A detection method, Western blot analysis of brain extracts from Lymnaea stagnalis (pond snail) and Poecilia reticulata (guppy) indicated the presence of four FMRFamide immunoreactive bands after a 7-day exposure to X-ray film. The most abundant peptide coelectrophoresed with the FMRFamide standard (M_r 598.8 Da), In addition, this Western blotting procedure also detected APGWamide (Ala-Pro-Gly-Try-NH₂; 428.5 Da) and [D-Ala²]-Leu-enkephalinamide (568.7 Da) with their respective specific antibodies.