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
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