2D-PAGE (IPG), Staining, Drying

First Dimensional Immobilized pH gradient (IPG)

1) Seat the tubes in the holes of the upper buffer reservoir of the tube cell. 
   Plug any unused hole with rubber stopper 
2) The lower chamber is filled with 0.02 N Sodium Hydroxide.
3) The water is removed from the top of the gel. 
   The samples are loaded in a volume 5 - 100 µl with a syringe and overlaid with 20 µl of 1/2 lysis buffer. 
   The upper chamber is filled with 0.02 N Phosphoric acid. 
   Connect positive terminal with lower chamber (acidic) and negative terminal to upper chamber (basic).  
   Connect positive terminal to lower chamber (basic) and negative terminal to upper chamber (acidic). 
   Run the current as follow
       400 V for  1 h
      1000 V for 16 h
      2000 V for  1 h

4) Remove the tubes and force the gels out onto a parafilm. 
   Wash each gel in 5ml 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 °C for several week or used immediately 


Second dimensional SDS-PAGE

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).