Protein Protocols Protein Protocols

Immunoblotting of 2-DE Separated Proteins 219
film may have different dimensions. Superimposing at the upper left corner for ECL
impression will allow subsequent matching of images.
4. Expose the film for a time variable from 5 s to several minutes. It is convenient to begin
with short exposure, develop the film, and then try longer exposures, if necessary.
5. Develop the film with the suitable reagents (see Note 16).
3.4. Stripping
At the end of a cycle of immunodetection, it is possible to strip the membrane with
indicated solution and to carry out subsequent cycles incubating with different primary
antibodies (see Note 18). The procedure for the stripping we use is:
1. Incubate the membrane in stripping buffer at 70°C for 30 min, with occasional shaking.
2. Wash the nitrocellulose 2 × 10 min in large volumes of washing solution at room
temperature.
3. Block the membrane and perform immunodetection as described in Subheading 3.3.
3.5. Matching
For an accurate matching process we use a computer program (see Note 19) that
permits matching the digitized images, using as landmarks the spots stained with
Ponceau S (see Note 20).
To perform this operation we suggest the following procedure (cf. Fig. 1):
1. Scan the ECL-developed film, the Ponceau S-stained nitrocellulose membrane, and the
silver-stained gel of the same sample with a computing densitometer with a sufficient
resolution (see Notes 21 and 22).
2. Rotate left–right the nitrocellulose membrane, with an appropriate program, in order to
have the three images with the cut lower corner on the right. In fact, the nitrocellulose
membrane has the spots only in one face and the scanning process generates an image
with the cut lower corner placed on the left.
3. Stack together the film and nitrocellulose membrane images, aligning the upper left corners
and the two corresponding borders and placing the cut lower right corner in the same
orientation for both.
4. Add “manually,” with appropriate software tool, the Ponceau S spots chosen as landmarks
onto the image of the ECL film.
5. Find the spots on the gel corresponding to landmarks on the film (see Note 23) and modify
the size of the silver nitrate image adjusting it to the smaller one of film by the mean of
adequate software. Actually the gel is larger than the film due to silver staining procedure.
6. Stack together the equalized ECL film and gel images, superimpose the landmarks carefully,
and run the automatic match program. This operation permits automatically highlighting
the silver-stained spots paired with the immunoreactive ones present on the
ECL film.
4. Notes
1. We perform electrotransfer from gels to nitrocellulose membranes by a “wet” procedure.
In a tank or “wet” apparatus the gel is submerged in a large volume of buffer during the
transfer. We use a Bio-Rad transfer cell with 3 L of transfer buffer or an ISODALT cell
(Hoefer Scientific Instruments) with 20 L of transfer buffer. ISODALT cells enable the
simultaneous transfer of five gels. The transfer buffer can be used several times, if stored
at 4°C.