How can western blots be visualized?

Western blots can be visualized using three primary detection methods:

Colorimetric Detection

In the colorimetric detection method, a substrate reacts with a reporter enzyme such as Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP), which is bound to the secondary antibody. As a result of this reaction, the soluble dye gets converted to a differently colored, insoluble product that appears as visible colored spots or bands on the membrane, which represent the target protein. 

This method is simple, fast, and can be analyzed visually without imaging equipment.   

Chemiluminescent Detection

Chemiluminescent detection uses a substrate that emits light instead of a colored precipitate when it reacts with Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP) reporter enzyme conjugates. This light emission is captured using an X-ray film or CCD digital imager. The captured image allows for qualitative and semi-quantitative analysis of the protein by densitometry.

Fluorescent Detection

Fluorescent detection uses a fluorescently labeled probe that emits light when excited by a specific wavelength of light. This emission is captured using a CCD camera equipped with appropriate emission filters or multichannel fluorescent scanners that enable qualitative and quantitative data to be obtained. 

This method allows for multiplex detection, so multiple proteins can be visualized on the same blot using a combination of fluorophores with varying excitation and emission spectra. Fluorescent detection is highly quantitative, but less sensitive than chemiluminescence.