Enhanced Chemiluminescent Detection

To detect the immunoreactive spot(s) with a chemiluminescent method it is necessary to wear gloves to prevent hand contact with film. We routinely use Enhanced Chemiluminescet detection (Amersham Biosciences) because it offers a greater sensitivity and the possibility of quantification of immune reaction (see Notes 17 and 20).

Mix equal volumes of detection reagent 1 and detection reagent 2 from the Amersham ECL kit (Subheading 2.2., step 14) and immerse the membrane in this solution for 1 min, ensuring that the surface of the membrane is completely covered with solution. The chemiluminescent signal can be acquired by a chemilumines-cence image acquisition system or by the following procedure:

1. Place the membrane on a glass and cover it with a layer of Saran Wrap®.

2. Cut away a corner from a piece of autoradiography film to define its orientation (see Subheading 3.1., step 10). Superimpose the autora-diography film on the nitrocellulose membrane beginning from the upper left corners. Nitrocellulose membrane and X-ray film may have different dimensions. Superimposing at the upper left corner for ECL impression will allow subsequent matching of images.

3. Expose the film for a time variable from 5 s to several minutes. It is usual to begin with a short exposure, followed by development of the film, followed by longer exposures, if necessary.

4. Develop the film with the suitable reagents (Subheading 2.2., step 17).

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