Immobilized pH gradient (IPG) as first dimension

protocol mainly based on http://www.expasy.ch/ch2d/protocols/

IPG gel strips rehydration

Hundred µg to 15 mg of proteins were solubilized with 350 µl of a solution containing 8 M urea, 4% CHAPS, 65 mM DTE, 0.8% resolytes 4-8 and a trace of bromophenol blue. To achieve reswelling and simultaneous loading of the sample, the entire samples were pipetted into the IPG strip holder,  grooves,  IPG strips (3.5-10 NL, 18 cm from Pharmacia Biotech.) were positioned such that the gel of the strip was in contact with the sample (up side down), and the gel and the sample were covered with 3 ml low viscosity paraffin oil to avoid evaporation.

Running conditions

Rehydration in the IPGphor for at least 10 hours, then the voltage was  increased from 200 to 8000 V.  Focusing was carried out for a total of 100 kVh. After running, strips can be frozen (-20^oC) for several weeks (remove oil), or used immediately for the second dimension.

IPG gel strips equilibration

After the first dimension run the strips were equilibrated in order to resolubilise the proteins and to reduce -S-S- bonds. The strips were equilibrated in custom-made tubes in a solution containing Tris-HCl (50 mM) pH 6.8, 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.

SDS-PAGE as second dimension

In second dimension, the Hoefer DALT system was used according to the user manual 80-6431-50 from amersham pharmacia biotech. Piperazine diacrylyl (PDA) is used as crosslinker. We believe this reduces N-teminal protein blockage, gives better protein resolution, and reduces diamine silver staining background.The gels were cast as described in the manual, with a linear gradient from 9 to 16.5% using the DALT multiple gel caster that allowed to cast simultaneously 23 gels.

IPG gel strips transfer

After the equilibration, the IPG gel strips were loaded onto the second dimension gels and then sealed with a solution containing 0.5% agarose in SDS Electrophoresis buffer at 70°C.

Running conditions

The gels were run at 15°C for 2 hours at 50 V, the voltage was then increased to max. 500 V and the gels run until the bromophenol blue front reaches the end of the gel.
  The proteins were then detected by silver staining.