Western Blotting Protocol
This protocol is designed for transferring proteins using the BioRad Transblot.
1. Chill transfer buffer at -20 ° C for at least one hour before use. Freeze ice-packs.
2. Soak gel, transfer pads, and Whatman 3MM paper in transfer buffer for 30-60 min at 4°C.
3. Assemble in cassette starting on black side: pad, filter, gel, pre-wetted nitrocellulose, filter, pad. Ensure there are no air bubbles.
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).
5. Remove NC filter. Place in seal-a-meal bag. Add 20 mL Blotto buffer. Shake 1 h at RT or 37°C (Can be left O/N at 4°C).
6. Discard blotto. Wash once with 20 mL PBS-TWEEN.
7. Add 20 mL blotto and 20 µL 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°C; or O/N at 4°C.
8. Remove blotto and antiserum. Add sodium azide to 0.02%. Store diluted antiserum at 4°C.
9. Wash filter with 20 mL PBS-TWEEN for 15′. Repeat for total of 3 times
10. Add 20 mL blotto and 20 µL of goat anti-rabbit-AP (Fisher). Incubate 1h at RT.
11. Repeat #9.
12. Rinse with 1xPBS. Then soak in 10 mL 1x alkaline phosphatase buffer for 15′.
13. Immediately before (<30 min) color development mix:
14. Develop blot until desired intensity is achieved.
15. Stop in STOP buffer for 15 min. Then place in water O/N.
16. Air dry, keep out of bright light.
Alkaline phosphatase buffer:
100 mM Tris-Cl pH 9.5
100 mM NaCl
5 mM MgCl2
10 mM Tris-Cl, pH 6.0
5 mM EDTA
NBT: (nitroblue tetrazolium)
50 mg/mL in 70% N<N,-dimethyl formamide
BCIP: (5-bromo-4-chloro-3-indolyl phosphate)
50 mg/mL in 70% formamide