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
- Blocking solution: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5% (w/v) skimmed milk powder, 2.5% normal serum (normal serum from the host-species of the secondary antibodies is recommended for blocking), 0.1% Tween 20 without sodium azide
- Incubation solution: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20, 5% (w/v) skimmed milk powder
- Washing solution A: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20
- Washing solution B: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl
- Substrate solution: Western Lightning® Plus-ECL PerkinElmer, Inc. or comparable product
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 blocking solution for 30 min on a lab shaker (gently rocking) at RT.
- Incubate in fresh incubation solution containing the primary antibody at the appropriate dilution and incubate for at least 2 h on a lab shaker at RT or overnight at 4°C.
- Wash 3 - 4 times with washing solution A for 10 min each time.
- Incubate in fresh incubation solution containing the recommended secondary antibody (anti-mouse IgG, anti-rabbit IgG, resp.) at the appropriate dilution and incubate for at least 1 h on a lab shaker at RT.
- Wash 3 times with washing solution A for 10 min each time.
- Replace washing solution A with washing solution B and let equilibrate for 5 min.
- Replace with fresh substrate solution and develop (X-ray film or ECL-reader). Exposure time can be shortened or extended, if signals are extremely strong or weak, resp.
A very weak signal may be caused by the primary and/or secondary antibody concentration being too high. The ECL substrate solution has a limited capacity and high amounts of local peroxidase can use up all the substrate within seconds before the picture is taken in your ECL reader.
Please try a lower concentration of primary and secondary antibodies in this case.
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.