Protein Protocols Protein Protocols

DIGE 193
2. Push the strip down until it is in firm contact with the top of the SDS gel. Add the agarose
until it barely covers the top of the strip. Make sure to add evenly from both sides and to
avoid air bubbles. Burst bubbles with gel loading pipettor tips after the agarose hardens.
Make sure to mark the second dimension gel with sample ID.
3. Electrophorese in the cold room. For a run of about 8–10 h, use 20 mA per gel at constant
voltage. For a run of about 16 h, use 8–10 mA per gel.
3.5. Image Acquisition and Analysis
The layout of the imaging system is vital for the success of DIGE. The technique has
unique requirements, so that all the hardware and most of the software for image acquisition
had to be built from scratch. Our system is experimental and transitory in nature.
At the time of the writing, the apparatus is under constant revision and development
and is not commercially available in its current form. Thus, rather than give the exact
minutiae of the image acquisition and analysis process, we list the nature and aim of
the operations that are performed to arrive at the final result. Hopefully, this will assist
those who are interested in either building or acquiring their own imaging system.
At the writing of this manuscript, commercially available DIGE imagers are just
beginning to be developed. For those who might consider either building or acquiring
an imager, we list the absolute minimum requirements that the hardware must meet:
1. In addition to the obvious requirement that the gel not physically move during imaging,
no changes in protein spot position due to optically induced deformations should occur
while switching between channels. Thus, a multiple bandpass emission filter must be
employed.
2. The imaging hardware needs to be sensitive enough to detect minimally labeled proteins.
Since a cooled, coupled charge device (CCD) had been used previously to image in gel
fluorescently labeled proteins (7), we decided to use a scientific grade CCD camera. CCD
cameras without cooling do not have the requisite sensitivity of low noise capabilities.
3. The imaging cabinet must be light-tight and illumination must be filtered through the
appropriate filter sets.
In the current incarnation of the DIGE imager, the gels are placed flat on a black
Plexiglas surface at the bottom of the cabinet. The camera is mounted vertically over
the gel at about 30 cm away. Illumination is provided by two halogen lamps with
fiberoptic leads mounted on top of the cabinet at ±60° incident angles to the bottom to
provide an even field of illumination. A schematic of the hardware is shown in Fig. 2.
The standard image acquired by the imager is a 4 × 4 cm square made up from 256 ×
256 pixels, each storing 65,000 gray levels as unsigned short integers.
3.5.1. Image Acquisition
1. After the second dimension run, the gel are removed from their cassettes and incubated in
destain solution for a minimum of 1 h. Gels are placed under either destain solution or in
1% acetic acid in H2O during imaging.
2. Two images are acquired from the central region of the gel, one with each excitation filter.
A few spot intensities are compared and used to normalize the acquisition times for the
two channels in tile mode. Tile mode is where the entire gel is imaged into a single file by
stitching together thirty 4 × 4 cm squares to generate one 20 × 24 cm image. Two such
images are generated, one for each channel. Each image corresponds to one of dyes and