This protocol is designed for transferring proteins using the\u00a0OWL Transblot.<\/strong><\/p>\n 1. Chill transfer buffer at -20 \u00b0 C for at least one hour before use.<\/p>\n 2. Soak gel, transfer pads, and Whatman 3MM paper in transfer buffer for 30-60 min at 4\u00b0C.<\/p>\n 3. Assemble the following in the cassette starting on black side: pad, filter, gel, pre-wetted nitrocellulose (NC), filter, pad. Ensure there are no air bubbles.<\/p>\n 4. Fill transfer assemble 3\/4 full with transfer buffer. Insert transfer cassettes. Run at constant 150 mA\u00a0 for 1.25 h<\/p>\n 5. Remove NC filter. Place in petri dish.. Add 10- 20 mL Blotto buffer. Shake 1 h at RT\u00a0 (Can be left O\/N at 4\u00b0C).<\/p>\n 6. Discard blotto. Wash once with 20 mL PBS-TWEEN.<\/p>\n 7. Add 10 – 20 mL blotto and 10-20 \u00b5L of antiserum (1:1000 dilution; this is a good starting place but the dilution value must be determined empirically for each antiserum). Incubate 1 h at RT or O\/N at 4\u00b0C.<\/p>\n 8. Remove blotto and antiserum. Add sodium azide to 0.02%. Store diluted antiserum at 4\u00b0C.<\/p>\n 9. Wash filter with 20 mL PBS-TWEEN for 15′. Repeat for total of 3 times<\/p>\n 10. Add 10 mL blotto and 10 \u00b5L of goat anti-rabbit-AP (Fisher). Incubate 1h at RT.<\/p>\n 11. Repeat #9.<\/p>\n 12. Rinse with 1xPBS. Then soak in 10 mL 1x alkaline phosphatase buffer for 15′.<\/p>\n 13. Immediately before (<30 min) color development mix:<\/p>\n <\/p>\n 14. Develop blot until desired intensity is achieved.<\/p>\n 15. Stop in STOP buffer for 15 min. Then place in water O\/N.<\/p>\n 16. Air dry, keep out of bright light.<\/p>\n <\/p>\n COLOR DEVELOPMENT:<\/p>\n Alkaline phosphatase buffer:<\/strong><\/p>\n 100 mM Tris-Cl pH 9.5<\/p>\n 100 mM NaCl<\/p>\n 5 mM MgCl2<\/p><\/blockquote>\n Stop buffer:<\/strong><\/p>\n 10 mM Tris-Cl, pH 6.0<\/p>\n 5 mM EDTA<\/p><\/blockquote>\n NBT: (nitroblue tetrazolium)<\/strong><\/p>\n 50 mg\/mL in 70% N:N,-dimethyl formamide<\/p><\/blockquote>\n BCIP: (5-bromo-4-chloro-3-indolyl phosphate)<\/strong><\/p>\n 50 mg\/mL in 100% formamide<\/p><\/blockquote>\n","protected":false},"excerpt":{"rendered":" Western Blotting Protocol This protocol is designed for transferring proteins using the\u00a0OWL Transblot. 1. Chill transfer buffer at -20 \u00b0 C for at least one hour before use. 2. Soak gel, transfer pads, and Whatman 3MM paper in transfer buffer …<\/p>\n