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Immobilized pH gradient (IPG) as first dimension

A non-linear immobilized pH gradient (3.5-10.0 NL IPG 18 cm) was used as the first dimension. It offered high resolution, great reproducibility and allowed high protein loads. Based on our specifications, the non-linear pH gradient strips were prepared by Pharmacia-Hoeffer Biotechnology AB and are commercially available. The strips were 3 mm wide and 180 mm long [6-12].

IPG gel strips rehydration

Hydration was performed overnight in the Pharmacia reswelling cassette with 25 ml of a solution containing urea (8 M), CHAPS (2% w/v), DTE (10 mM), Resolyte pH 3.5-10 (2% v/v) and a trace of Bromophenol Blue [6].

Sample application

When the rehydration cassette had been thoroughly emptied and opened, the strips were transferred to the Pharmacia strip tray. After placing IPG strips, humid electrode wicks, electrodes and sample cups in position, the strips and cups were covered with low viscosity paraffin oil. Samples were applied at the cathodic end of the IPG strips in a slow and continuous manner, without touching the gels [6].

Running conditions

The voltage was linearly increased from 300 to 3500 V during 3 hours, followed by 3 additional hours at 3500 V, whereupon the voltage was increased to 5000 V. A total volthourproduct of 100 kvh was used in an overnight run [6].

IPG gel strips equilibration

After the first dimension run the strips were equilibrated in order to resolubilize the proteins and to reduce -S-S- bonds. The strips were equilibrated within the strip tray with 100 ml of a solution containing Tris-HCl (50 mM) pH 8.4, urea (6 M), glycerol (30% v/v), SDS (2% w/v) and DTE (2% w/v) for 12 min. -SH groups were subsequently blocked with 100 ml of a solution containing Tris-HCl (50 mM) pH 6.8, urea (6 M), glycerol (30% v/v), SDS (2% w/v), iodoacetamide (2.5% w/v) and a trace of Bromophenol Blue for 5 min [6].

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