Protein Protocols Protein Protocols

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Fig. 2. DIGE Imager.
thus represents one of the samples which were run on the gel. Acquisition times typically
vary between 10 and 180 s per square, which translates to 10–180 min total acquisition
time per gels.
3.5.2. Image Analysis
1. The two images from a single gel are inverted and then normalized to each other so that
the most abundant spots appear at the same level of intensity. The images are then converted
to byte format and placed into a two-frame quicktime movie. Playing this movie in
a continuous loop allows for the visual detection of differences. The images are normalized
at several different grayscale levels. Normalizing for high values allows for the
detection of the abundant protein changes and normalizing at lower values covers the
lower-abundant protein changes.
4. Notes
1. A mixture of urea and thiourea has been reported to aid in the solubilization of membrane
proteins (8).
2. Never warm a protein lysate in urea; always endeavour to keep at least on ice, if not
frozen. Minimize the time samples spend away from –80°C. This is because at high
temperature and pH urea spontaneously breaks down to yield cyanate, which modifies
lysine residues and leads to carbamylated charge trains in the IEF dimension. Low
temperature slows down but does not stop this process (9).
3. Usually the pH of HEPES is adjusted with KOH; however, if SDS is used in any
subsequent step (such as running lysate directly on SDS-PAGE), KDS will be formed,
which is insoluble in water. Use NaOH. When making up this buffer, make sure to add the
HEPES last, see Note 2.