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 micron). 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 microgram 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.
Too
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Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada.
Murphy
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PR
Croll
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RP
D000818:Animals; D000906:Antibodies; D015153:Blotting, Western / Q000379:methods*; D001923:Brain Chemistry; D002854:Chromatography, Paper / Q000379:methods*; D003101:Collodion; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000032:analysis; D019835:FMRFamide; D005557:Formaldehyde; D017952:Ganglia, Invertebrate / Q000737:chemistry; D005780:Gelatin; D007457:Iodine Radioisotopes; D008195:Lymnaea; D008970:Molecular Weight; D009479:Neuropeptides / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D018377:Neurotransmitter Agents / Q000032:analysis; D010209:Paper; D011036:Poecilia; D011817:Rabbits / Q000276:immunology; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A
D000906:Antibodies; D007457:Iodine Radioisotopes; D009479:Neuropeptides; D018377:Neurotransmitter Agents; D013205:Staphylococcal Protein A; C063193:alanyl-prolyl-glycyl-tryptophanamide; D005557:Formaldehyde; D004743:Enkephalin, Leucine; C048614:enkephalinamide-Leu; D019835:FMRFamide; D005780:Gelatin; D003101:Collodion