4-Chloro-1-Naphthol Solution Protocol for colorimetric detection of western blot proteins

Product No. C8302

Product Description

Solution contains 0.48 mM 4-Chloro-1-Naphthol, 50 mM Tris-HCl and 0.2 M NaCl in 17% methanol. This is a substrate solution designed for visualizing horseradish peroxidase conjugates in Western Blotting.


Storage Temperature 2-8 °C

Reagents and Equipment Required but Not Provided

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. 


  1. After the gel is transferred onto a blotting membrane, wash the membrane for 5 minutes with the washing solution.
  2. Incubate the rinsed membrane with primary antibody diluted in blocking solution.  A blocking step prior to step 2 is usually not necessary.
  3. Wash the membrane for 5 minutes with the washing solution.
  4. Incubate the washed membrane with secondary antibody peroxidase conjugate in blocking solution for 2 hours at room temperature with gentle agitation. (A 1:1000 dilution of antibody is recommended to start).
  5. Wash the membrane 3 times for 5 minutes each in washing solution.
  6. Add hydrogen peroxide (H1009) to 4-Chloro-1-Naphthol Solution (C8302) to obtain a final concentration of 0.01% hydrogen peroxide (v/v). Prepare immediately before use.
  7. Cover the membrane with the substrate solution for 1-5 minutes at room temperature until the desired color is obtained. Use 10-20 ml for a 8x10 cm membrane.
    NOTE: Make sure the membrane is completely covered in solution.
  8. The color development can be stopped by extensive washing with water.
    NOTE: Watch carefully to prevent over-development of the signal.




  1. B. Batteiger, Journal of Immunological Methods, 55 (1982) 297-307.
  2. D.I. Scott, Journal of Immunological Methods, 119 (1989) 153-187.
  3. F. Miescher, Analytical Biochemistry, 119 (1982) 142-147.


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