Important: Some proteins have special requirements for good separation (e.g. unboiled samples or special gel systems). Please refer to the remarks sections for western blotting on the respective data sheet.
- Ponceau S staining solution: 5% acetic acid, 0.1% Ponceau S
- 5% skimmed milk-TBST: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5% (w/v) skimmed milk powder, 0.02% sodium azide, 0.1% Tween 20
- 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 10 ml. Prepare this solution shortly before use.
Separate the protein sample to be examined and a molecular weight standard using SDS-PAGE and transfer to a nitrocellulose membrane by electro-blotting. Follow the manufacturer's instructions for your SDS-PAGE and blotting device.
- Stain the membrane with Ponceau S staining solution for several minutes at room temperature to check the efficiency of transfer.
- Rinse the membrane in water to remove the Ponceau S staining solution and incubate in 5% skimmed milk-TBST for 30 min on a lab shaker at RT.
- Incubate in fresh 5% skimmed milk-TBST containing the primary antibody at the appropriate dilution for at least 2 h on a lab shaker at RT or over-night at 4°C.
- Wash 3 - 4 times with 5% skimmed milk-TBST for 10 min each time.
- Incubate with fresh 5% skimmed milk-TBST containing the recommended AP-conjugated secondary antibody (anti-mouse IgG, anti-rabbit IgG, resp.) at the appropriate dilution for at least 1 h on a lab shaker.
- Wash 3 times with 5% skimmed milk-TBST for 10 min each time.
- Wash with substrate buffer and 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.
Note: The SYSY standard protocol generates good results in the SYSY labs and may be used as a reference. However, to achieve the highest specific signal and lowest non-specific background signal, the best antibody concentration, incubation temperature, and incubation time must be determined individually.