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

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. 

Procedure

  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.

Materials

     

 References

  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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