First Dimensional Isoelectric Focussing (IEF) (Fig.1-1)
1) Mark 2cm from the top of clean, dry 13 cm long and 3 mm diameter, 1d gel tubes to indicate the desired height of the gel.
Place a rubber band around them so that they form a tight bundle. Hold the bundle vertically on a flat surface
2) Seal the bottom with two layers of parafilm
3) Prepare the solution
Urea 4.8 g
30% acrylamide stock 1.16 ml
NP-40 2 ml
Water 2.84 ml
Ampholines (pH5-7) 0.25 ml
Ampholines (pH 3.5 -10) 0.25 ml
10% APS 15 µl
TEMED 10 µl
Note. Dissolve the urea by swirling in water bath whose temperature should not higher than 60 °C
4) Using a long narrow guage hypodermic needle, fill the gel tubes with the solution of step no 3 to the mark.
5) Overlay the gel mix with 20 µl of water and allow it to polymerize it for one hour
6) Remove the para-film from the bottom of the tubes and water is removed from the top of the gel.
7) Seat the tubes in the holes of the upper buffer reservoir of the tube cell.
Plug any unused hole with rubber stopper
8) The lower chamber is filled with 0.02 N Phosphoric acid .
The samples are loaded in a volume 5 - 100 µl and overlaid with 20 µl of 1/2 lysis buffer.
The upper chamber is filled with 0.02 N Sodium Hydroxide.
9) Run the current as follows
1. 200 V for 30 minutes
2. 400 V for 16-18 hours
3. 600 V for 1 hour
10) Extrude the gel from the tubes using the water pressure from a syringe.
Wash each gel in 5 ml SDS sample buffer for 15 minutes. Change the buffer and wash it again for 15 minutes.
At this point the gel can be stored at -20 oC for several week or used immediately.
Second dimensional SDS-PAGE (Fig.1-2)
1) Assembled detergent cleaned gel plates.
The separating gel is cast first, followed by the casting of the upper stacking gel.
For a gel with dimension 1 mm x 16cm x 14cm, about 20 ml of separating gel mix and about 5ml of stacking gel mix is needed.
2) Prepare 15 % polyacrylamide gel as shown below
Acryalamide for separating gel 8.5 ml
Separating gel buffer (pH.8.8) 6.3 ml
Water 2.0 ml
10% APS 120 µl
EMED 20 µl
For one plate
Swirl to mix and pour the gel immediately. Gently overlay with 1cm of water.
Allow the gel to polymerize for 45 to 60 minutes
3) Preparation of Stacking gel solution
Stacking gel acrylamide 1.0 ml
Stacking gel buffer 3.0 ml
Water 2.0 ml
APS 30 µl
TEMED 20 µl
Remove the overlaid water and pour the stacking gel solution immediately.
Leave it to polymerize for 10-15 minutes
4) The first dimension is applied directly on the top of the stacking gel. The first dimension is overlaid by 1% agarose.
5) The slab gel is assembled for running. A few drops of tracking dye (bromophenol blue) is added
6) Run the sample at 35 mA (constant current) until the tracking dye reaches the bottom of the separating gel.
7) Separate the gel plates and either stain the gel or process for western blot.
The gel is stained either with silver staining or Coomassie brilliant blue(CBB).
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