Protein Protocols Protein Protocols

Western Blotting Using ECL 433
Fig. 1. Western blots of actin dilutions detected with ECL Plus. Actin concentrations from
the left: 400, 200, 150, 100, 75, 50, 35, 25, 12.5, 6.25 ng, blank, “unknown.” The “unknown”
was a test sample whose concentration was calculated from the standard curve. (A) Film autoradiograph,
exposure time 5 min. The film was digitized on a Personal Densitometer SI (PDSI).
(B) Fluorescence scanner (Storm, Model 860) image (same blot as in a). (C) CCD camera
(ImageMaster VDS-CL) image, 5 min exposure.
est concentration is only just visible should have the rest of the standards in the linear
range of the film.
The resultant film is then digitized with a densitometer and saved as a 12 bit file. The
band intensities can then be quantified using an appropriate image analysis program.
3.4. Detection Using a Fluorescence Scanner (See Fig. 2)
1. Ensure that the plastic used to encase the blot does not exhibit high fluorescence.
2. Blots should be scanned between 5 min and 1 h after addition of ECL Plus substrate
reagents.
3. The blot is scanned face down at 450 nm on the Storm scanner using blue fluorescence/
chemifluorescence scan mode. Set the photomultiplier tube (PMT) voltage between 500
and 900 for maximum linearity (see Note 4).
4. The band intensities are then quantified using an appropriate image analysis program such
as ImageQuant.
3.5. Detection Using a CCD Camera (See Table 1)
1. Exposure times will typically be between 30 s and 10 min depending on the sensitivity
desired and the type of camera used (see Note 5).