Bio-Rad Model 111 Mini IEF Cell - Manual (Page 16)

Section 5
Sample Detection and Gel Storage
5.1 Removing the Gel
1. After electrofocusing is complete, turn off the power supply and unplug the power cables.
2. Slide the protective lid from the cell and remove the gel from the electrodes. At this point
separate the gel/gel support film from the glass plate.
3. Remove the Graphite Electrodes and clean with water and a tissue. Rinse the chamber with
distilled water. Put the electrodes back into the cell to prevent damage during storage.
Cleaning the electrodes immediately after the run will increase their life span.
5.2 Band Detection
In general, proteins are detected by fixing and staining. If the proteins are not fixed
immediately, the high resolving power of electrofocusing can be lost to diffusion. Small proteins
and proteins with basic pIs are particularly difficult to fix. Autoradiography, biological activity,
group specific staining, and immunological methods are a few examples of alternative detection
techniques currently being used.
This section gives two recommended staining procedures for electrofocusing using Coomassie
blue R-250, which has a detection limit for most proteins in µg quantities. Either method produces
acceptable results, however, Method B is significantly easier to use and, in some cases, will
produce a sharper pattern. For more sensitive detection down to ng levels of protein, the Bio-Rad
Silver Stain (catalog number 161-0443) is recommended.
The cupric sulfate in the staining and destaining solutions effectively eliminates any
background staining due to the presence of ampholytes.
Fixing and Staining
Method A Method B
Fixative: Fixative:
4% sulfosalicylic acid Not necessary
12.5% trichloroacetic acid
30% methanol
Immerse gels in this solution for 30 minutes.
Method A Method B
Stain: Stain:
27% isopropanol or ethanol Same as Method A, but with
10% acetic acid 0.05% crocein scarlet
0.04% Coomassie brilliant blue R-250
0.5% CuSO4
(0.05% crocein scarlet optional)
Dissolve the CuSO4 in water before adding the alcohol.
Either dissolve the dye in alcohol or add it to the solution
at the end.
Immerse the gel in the stain for approximately 1-2 hours.
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Crocein scarlet, a highly soluble
dye which rapidly binds to
protein, is included to assure rapid
fixation of the bands. This
procedure is inadequate if
Coomassie brilliant blue R-250 is
used alone.