Thesis final - after defense-7 - Jacobs University

Chapter 2
IPG strips were kept for 16 hours in rehydration buffer (6 M urea, 2 M thiourea, 1% CHAPS,
0.4% DTT, and 0.5% v/v pharmalyte TM 3-10). The sample 200 µl (65 µg proteins in 75 + 125
µl of rehydration buffer) was applied in in-gel rehydration buffer instead of cup loading. The
sample was allowed to absorb on a strip for 15 min, and then covered with silicon oil. After a
sixteen hour rehydration period, focussing was performed. IEF was performed on these
conditions: 50 V, 120 min. 150 V, 90 min. 500 V, 60 min, 1500 V, 40 min. 2500 V, 40 min
and 3500 V, 180 min at 20°C. The minute amount of buffer ions present in the samples was
removed by applying low voltage (100 volts) at the beginning for 5 hours and the filter paper
beneath the electrode (where the salt ions have collected) was changed time by time. After
focussing, the strips were first equilibrated for 15 minutes in 10 ml equilibration buffer (50
mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% w/v SDS and 10 mg/ml DTT) containing
20 µl bromophenol blue and subsequently for additional 15 minutes in the same buffer
containing 25 mg/ml iodoacetamide instead of DTT. After equilibration, the strips were kept
on top of a 12.5% SDS vertical slab and covered by 0.7% hot low melting point agarose in
SDS electrophoresis running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). The sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a Hoefer
mini VE vertical electrophoresis system in 1 mm thick 10 cm × 10.5 cm gels, 25 mA per gel
and 120 volts (19). Colloidal coomassie blue was used for staining the gels (89). The gels
were first fixed for at least three hours in the fixing solution (50% ethanol and 3% phosphoric
acid). The gels tend to shrink during fixing. Then the gels were washed in deionized water
three times for 20 minutes at a time. After washing, the gels were preincubated for 1 hour in
staining solution (34% methanol, 3% phosphoric acid and 17% w/v ammonium sulfate). Then
a small amount of coomassie blue was added with a spatula tip to the gels and stained
overnight. After staining, the gels were washed with deionized water to remove the
background stain. The gels were scanned by 48 bit Epson color scanner (Epson perfection
4990 Photo). 2-D gels of the chromatography fractions were performed in duplicate and the
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