Ipgphor system

For 1D gel electrophoresis, 20 µg pollen extract solubilized in denaturing/reducing sample buffer was applied to each well of 12% SDS-PAGE. In 2D electrophoresis, for the isoelectric focusing (IEF), the proteins were solubilized in a rehydration solution (DeStreak plus 0.5% IPG buffer (GE Healthcare Biosciences AB, Uppsala, Sweden) and bromophenol blue). Immobiline Dry Strips (IPG) with a pI range of 3–10 or 4–7 were rehydrated in this protein solution for 15 h, aiming at a good separation of proteins from different pIs probably encountered in the extract. The strips were submitted to IEF in an IPGphor system (GE Healthcare Biosciences AB, Uppsala, Sweden) at 300, 1000 and 5000 V until reaching 5750 Vh. For reduction and alkylation, two steps were performed, first with 1% DTT for 15 min, followed by 4% iodoacetamide (IAA) for 15 min, before the strips were loaded onto 12% polyacrylamide gels for protein separation by reducing SDS-PAGE. Proteins were stained with Coomassie Blue Colloidal [41 (link)], and gel images were captured using ImageQuant LAS 4000 (GE Healthcare Biosciences AB, Uppsala, Sweden).

The two dimensional-differential gel electrophoresis (2D–DIGE) procedure was performed as previously described [18 (link)]. Briefly, soluble adipose proteins (50 μg) were minimally labelled with 400 pmol of amine-reactive Cy3 or Cy5 fluorescent cyanine dyes (GE Healthcare, Saclay, France) and an internal standard comprising equal amounts of each protein extract was labelled with Cy2 dye. For each gel, two samples were run with the pooled standard (Additional file 1) in 24 cm precast immobilized pH 4–7 gradient strips and using an IPGphor system (GE Healthcare). Isoelectric conditions were optimized to obtain well-resolved protein spots in 2D–gels at both ages: stepwise voltage increase with 120 V for 1 h, 200 V for 1 h, 500 V for 1 h, 1,000 V for 6 h, linear gradient up to 8,000 V within 1.5 h, and 8,000 V for 7.5 h for a total focusing of 74,300 V-h. Proteins were then separated on 12.5% SDS polyacrylamide gels using a vertical Ettan Dalt Six device (GE Healthcare). Two preparative gels (one for d 90 and one for d 110) made with 600 μg of proteins from the corresponding pooled extracts were run in the same conditions and stained with LavaPurple (Serva Electrophoresis, CliniSciences, Nanterre, France). Representative 2D–gels of fetal adipose tissue obtained at d 90 or d 110 of gestation were shown in Additional file 2.

Immobiline DryStrips, linear pH 3–10, 7 cm long (Amersham Biosciences, Uppsala, Sweden) were rehydrated overnight at 25°C with 250 μg of protein in 125 μL of rehydration solution (8 M urea, 2% CHAPS, 0.2% DTT, 0.5% IPG Buffer pH 3–10, 0.002% bromophenol blue) for each treatment. After rehydration, the first-dimension isoelectric focusing (IEF) was performed using the IPGphor system (GE Healthcare). The proteins were separated at 20°C using 50 μA per strip with the following linear voltage increase: 200 V for 2 h, 1000 V for 1 h, and 5000 V for 1.5 h. The strips were then incubated in equilibration buffer [50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS] containing 0.5% (w/v) dithiothreitol (DTT) for 15 min, followed by equilibration buffer containing 4% (w/v) iodoacetamide (IAA) for 15 min.
The IPG strip was then transferred onto SDS-PAGE [12.5% (w/v) polyacrylamide and 0.8% (w/v) bis-(N,N’-methylenebisacrylamide)] and the second dimension was performed at 10 mA/gel for 15 min and 20 mA/gel for 1 h at 10°C in a Mini-VE system (GE Healthcare). The gels were stained overnight with Coomassie Brilliant Blue R-250 (CBB) and stored at 21°C in preserving solution [7% (v/v) acetic acid].