Standard protocol for Western blots with alkaline phosphatase staining
We use this protocol for the routine testing of our antibodies. It has turned out to be very robust and suitable for almost all our antibodies.
- Blot buffer: 192 mM glycine; 25 mM Tris-base; 20 % (v/v) methanol; 0.04 % SDS.
- Ponceau S staining solution: 5 % acetic acid, 0.1 % Ponceau S.
- Blocking solution: 20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 5 % (w/v) skimmed milk powder; 5 % normal serum (normal serum from the host-species of the secondary antibodies is recommended for blocking); 0.02 % sodium azide; 0.1 % Tween 20.
- Washing solution: 20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.1 % Tween 20; 5 % (w/v) skimmed milk powder.
- Substrat buffer for alkaline phosphatase: 100 mM Tris-HCl, pH 9.5; 100 mM NaCl; 5 mM MgCl2
- BCIP staining solution: 20 mg/ml in 100 % di-methyl formamide.
- NBT staining solution: 50 mg/ml in 70 % di-methyl formamide.
- Staining solution complete: Substrate buffer containing 80 µl BCIP solution and 60 µl NBT solution per 10ml and is mixed freshly for each staining.
The protein sample to be examined is separated by SDS PAGE and transferred to a nitrocellulose membrane by semi-dry electro-blotting using ~0.8 mA/cm2 (~0.3 mA/inch2).
- Stain the membrane with Ponceau S staining solution for several minutes at room temperature to check transfer.
- Rinse the membrane in water and incubate in blocking solution for 30 min on a lab shaker (gently rocking).
- Replace with fresh blocking solution containing the primary antibody in the appropriate dilution and incubate for at least 2 h on a lab shaker.
- Wash 3 - 4 times with washing solution for 10 min each time.
- Replace with fresh washing solution containing the recommended secondary antibody (anti-mouse IgG, anti-rabbit IgG, resp.) in the appropriate dilution and incubate for at least 1 h on a lab shaker.
- Wash 3 times with washing solution for 10 min each time.
- Replace washing solution with substrate buffer and let equilibrate for 5 min.
- Replace with fresh staining solution complete and develop for 15 - 30 min. Time can be shortened or extended, if signals are extremely strong or weak, resp.
- Stop staining reaction by washing 3 times with H2O.