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5.4 Isoelectric
focusing
using
Ampholine
PAGplate
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This section describes the running procedure for IEF using Ampholine
PAGplate. For further information, see the instructions supplied with the
product.
Switch on MultiTemp II thermostatic circulator and set the temperature
(normally 10 °C for polyacrylamide IEF) 15 minutes before starting the
experiment.
Cover the holes in the safety lid with tape to limit the amount of CO
contact with the gel, thereby improving the basic region of the pH gradient.
If desired, 100 ml of 1 M sodium hydroxide solution may be poured into
the buffer chambers to absorb CO
Make up about 100 mls of the required electrode solutions. (See Table).
Electrode solutions for IEF using Ampholine PAGplate
pH range
Anode solution
3.5-9.5
1 M Phosphoric acid
4.0-6.5
0.1 M Glutamic acid
5.5-8.5
0.4 M HEPES
4.0-5.0
1 M Phosphoric acid
5.0-6.5
0.01 M Acetic acid
Note: Wear clean gloves when working with polyacrylamide gels.
Pipette 1 ml of insulating fluid (kerosene or light paraffin oil) onto the
cooling plate. Open one package of Ampholine PAGplate and position the
gel with the stiff plastic film facing down on the cooling plate. Make sure no
air bubbles are trapped under the gel. Use the screened template on the
cooling plate to centre the gel. Remove any excess solution with a paper
tissue.
Soak the electrode strips evenly in the appropriate electrode solution (see
table above), approximately 3 ml/strip. The surface of the strips should look
wet. Remove excess solution with a tissue paper.
and further improve gradient stability.
2
Cathode solution
1.0 M NaOH
0.1 M ß-alanine
0.1 M NaOH
1.0 M Glycine
0.01 M NaOH
5. Operation
in
2
31
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