Colorimetric Detection of Western blot using 4-Chloro-1-Naphthol Solution Protocol

Product Description

4-Chloro-1-Naphthol solution (Product No. C8302) contains 0.48 mM 4-Chloro-1-Naphthol, 50 mM Tris-HCl and 0.2 M NaCl in 17% methanol. This solution is used as a substrate for detecting horseradish-peroxidase conjugates in Western blotting.

Storage

Store at 2-8 °C

Additional Reagents Required

  • 10X Tris-buffered saline (TBS) (Product No. T5912)
  • 20% TWEEN® 20 detergent (Product No. P9203)
  • Bovine serum albumin (BSA) Fraction V powder (Product No. A9647)
  • Nonfat dry milk (Product No. M7409)
  • Hydrogen peroxide (Product No. H1009)

Precautions and Disclaimer

This product is for research 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 wash buffer (TBS).
  2. Incubate the rinsed membrane with primary antibody diluted in blocking solution (1-3% BSA or nonfat dry milk in TBS). A blocking step before 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 (Product No. H1009) to 4-Chloro-1-Naphthol Solution (Product No. 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
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References

1.
Batteiger B, Newhall V WJ, Jones RB. 1982. The use of tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. Journal of Immunological Methods. 55(3):297-307. http://dx.doi.org/10.1016/0022-1759(82)90089-8
2.
Stott D. 1989. Immunoblotting and dot blotting. Journal of Immunological Methods. 119(2):153-187. http://dx.doi.org/10.1016/0022-1759(89)90394-3
3.
Hawkes R, Niday E, Gordon J. 1982. A dot-immunobinding assay for monoclonal and other antibodies. Analytical Biochemistry. 119(1):142-147. http://dx.doi.org/10.1016/0003-2697(82)90677-7