Western Blotting of Formaldehyde-Fixed Neuropeptides as Small as 400 Daltons on Gelatin-Coated Nitrocellulose Paper
✍ Scribed by C.K.L. Too; P.R. Murphy; R.P. Croll
- Book ID
- 102965123
- Publisher
- Elsevier Science
- Year
- 1994
- Tongue
- English
- Weight
- 787 KB
- Volume
- 219
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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 \mu \mathrm{m})). The electroblotted peptides were fixed to the nitrocellulose paper for 5-10 min in (4 %) paraformaldehyde solution. Using anti-rabbit FMRFamide (Phe-Met-Arg-Phe- (\mathbf{N H}{2}) ) as primary antibody, positive immunoreactivity was detected with an amplified alkaline phosphatase assay which was sensitive to at least (0.5 \mu \mathrm{g}) FMRFamide/lane. When immunoreactivity was determined with ({ }^{125}) I-protein A, it was possible to amplify and detect weak signals by increasing the autoradiography time. Therefore, using the ({ }^{125} I)-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{\mathbf{r}} 598.8) Da). In addition, this Western blotting procedure also detected APGWamide (Ala-Pro-Gly-Try-NH (\mathbf{N}_{2} ; 428.5) Da) and [D-Ala (\left.{ }^{2}\right])-Leu-enkephalinamide ( 568.7 Da) with their respective specific antibodies. § 1994 Academic Press, Inc.