5 Equilibration of the IPG gel strips

The IPG gel strips are equilibrated twice, each time for 15 min in 2x10 ml equilibration buffer (Fig. 13). The equilibration buffer contains 6 M urea and 30% glycerol in order to diminish electroendosmotic effects (Görg et al. 1988) which are held responsible for reduced protein transfer from the first to the second dimension. During the 2nd equilibration step, 260 mM iodoacetamide is added to the equilibration buffer in order to remove ecxess DTT (responsible for the "point streaking" in silver stained patterns) (Görg et al. 1987). The equilibrated IPG gel strips are slightly rinsed and blotted to remove ecxess equilibration buffer and then applied onto the second dimension SDS gel. Alternatively, TBB may be used instead of DTT and IAA (Herbert et al. 1997).

Note: Shorter equilibration times can be applied, however at the risk that some proteins may not migrate out of the IPG gel strip during sample entry into the SDS-PAGE. In this case it is advisable to check, by staining the IPG strip after removal from the SDS gel, whether all proteins have left the IPG strip.

Sodium dodecyl sulfate (SDS) (Serva), iodoacetamide, D,L-dithiothreitol (DTT) tris(hydroxymethyl)aminomethane (Tris) (Sigma), bromophenol blue, glycerol, urea, sodium azide (Merck)

Resolving gel buffer: 1.5 M Tris-HCl, pH 8.8 and 0.4% (w/v) SDS (Laemmli 1970).

To make 100 ml , dissolve 18.2 g of Trizma base and 0.4 g of SDS in about 80 ml of deionized water. Adjust to pH 8.8 with 4N HCl and fill up to 100 ml with deionized water. Add 10 mg of sodium azide and filter. The buffer can be stored at 4°C up to 2 weeks.

Equilibration buffer: 6 M urea, 30% (w/v) glycerol and 2% (w/v) SDS in 0.05 M Tris-HCl buffer, pH 8.8. To make 500 ml add: 180 g of urea, 150 g of glycerol, 10 g of SDS and 16.7 ml of resolving gel buffer. Dissolve in deionized water and fill up to 500 ml. The buffer can be stored at room temperature up to 2 weeks.

Bromophenol Blue solution: 0.25% (w/v) of Bromophenol blue in resolving gel buffer. To make 10 ml: dissolve 25 mg of Bromophenol blue in 10 ml of resolving gel buffer. Store at 4°C.

1. Dissolve 100 mg of DTT in 10 ml of equilibration buffer (= equilibration buffer I). Take out the focused IPG gel strips from the freezer and place them into individual test tubes (Fig. 13). Add 10 ml of equilibration buffer I and 50 µl of the Bromophenol Blue solution. Seal the test tubes with Parafilm, rock them 15 min on a shaker and then pour off equilibration buffer I.

2. Dissolve 400 mg of iodoacetamide in 10 ml of equilibration buffer (= equilibration buffer II). Add equilibration buffer II and 50 µl of Bromophenol Blue solution to the test tube as above and equilibrate for another 15 min on a rocker.

3. After the 2nd equilibration, rinse the IPG gel strip with deionized water for a second and place it on a piece of filter paper at one edge for a few minutes to drain off ecxess equilibration buffer.