Isoelectric Focusing Using Cleangel Ief - Pharmacia Biotech Multiphor II User Manual

5.5 Isoelectric
focusing
using
CleanGel IEF
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Detection methods
For automated silver and coomassie staining of polyacrylamaida gels see
Protocol guide, Hoefer Automated Gel Stainer (80-6343-34).
PhastGel Blue staining
On completion of isoelectric focusing, remove the electrode strips from the
gel. Immerse the gel in 250 ml of solution according to the schedule below
(See section 4.9 for stock solutions). The staining and destaining steps
should be carried out on a shaking table.
Step No. Solution
1 Fixing solution N
2 Destaining solution I
3 Staining solution K
4 Destaining solution I
5 Destaining solution I
6 Preserving solution O
The staining solution should be heated to 60 °C and poured over the gel.
No further heating is necessary. Destain the gel using several changes of
destaining solution (I) until the background is clear. Change the solution
frequently (particularly at the beginning) in order to speed up the destaining.
To preserve the gel, soak a cellophane sheet in preserving solution (I) and
lay it on the gel surface. Remove any air bubbles and wrap the excess
cellophane around the glass plate. An additional glass plate may be placed
underneath during drying to stop the cellophane from shifting or wrinkling.
Leave the gel at room temperature until it is completely dry.
CleanGel IEF is a washed and dried polyacrylamide gel optimized for
analytical isoelectric focusing.
Prior to use, the dried gel is rehydrated in a solution containing Ampholine
or/and Pharmalyte. Additives such as urea and/or detergents can also be
added at this stage. CleanGel IEF is rehydrated in a flat tray (GelPool) to a
thickness of 0.43 mm. Instructions are supplied with the product.
Time (min) Temp (°C)
20 23
2 23
10 60
20 23
30 23
10 23
5. Operation
35