Transfer to nitrocellulose and alkaline phosphatase detection (BioRad)

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:


10 mL AP buffer + 66 µL NBT + 33 µL BCIP

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

Stop buffer:

10 mM Tris-Cl, pH 6.0


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