This protocol is designed for transferring proteins using the\u00a0BioRad Transblot.<\/strong><\/p>\n 1. Chill transfer buffer at -20 \u00b0 C for at least one hour before use. Freeze ice-packs.<\/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 in cassette starting on black side: pad, filter, gel, pre-wetted nitrocellulose, 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 100 V for 1.25 h (130-180 mA).<\/p>\n 5. Remove NC filter. Place in seal-a-meal bag. Add 20 mL Blotto buffer. Shake 1 h at RT or 37\u00b0C (Can be left O\/N at 4\u00b0C).<\/p>\n 6. Discard blotto. Wash once with 20 mL PBS-TWEEN.<\/p>\n 7. Add 20 mL blotto and 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 3h at RT; 1.5h at 37\u00b0C; 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 20 mL blotto and 20 \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:<\/strong>\u00a0(nitroblue tetrazolium)<\/p>\n 50 mg\/mL in 70% N<N,-dimethyl formamide<\/p><\/blockquote>\n BCIP:<\/strong>\u00a0(5-bromo-4-chloro-3-indolyl phosphate)<\/p>\n 50 mg\/mL in 70% formamide<\/p><\/blockquote>\n","protected":false},"excerpt":{"rendered":" Western Blotting Protocol This protocol is designed for transferring proteins using the\u00a0BioRad Transblot. 1. Chill transfer buffer at -20 \u00b0 C for at least one hour before use. Freeze ice-packs. 2. Soak gel, transfer pads, and Whatman 3MM paper in …<\/p>\n