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Standard Protocol for Western Blots with Horseradish Peroxidase Detection

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.

Solutions needed

  • 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.

  1. Stain the membrane with Ponceau S staining solution for several minutes at room temperature to check the efficiency of transfer.
  2. 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.
  3. 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.
  4. Wash 3 - 4 times with washing solution A for 10 min each time.
  5. 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.
  6. Wash 3 times with washing solution A for 10 min each time.
  7. Replace washing solution A with washing solution B and let equilibrate for 5 min.
  8. 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.