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SDS-PAGE as second dimension

 In second dimension, a vertical gradient slab gel with the Laemmli-SDS-discontinuous system was used with some small modifications [13-15].

  1. Gels are not polymerized in the presence of SDS. This seems to prevent the formation of micelles which contain acrylamide monomer, thus increasing the homogeneity of pore size and reducing the concentration of unpolymerized monomer in the polyacrylamide. The SDS used in the gel running buffer is sufficient to maintain the necessary negative charge on proteins.
  2. 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.
  3. Sodium thiosulfate is used as an additive to reduce background in the silver staining of gels.
  4. The combination of the IPG strip and agarose avoids the need for a stacking gel.

In addition, the gels were cast with the Angelique system from LargeScaleBiology, which it is an efficient and easy to use PC control equipment that allowed to cast simultaneously 10 to 60 gels.

Gel composition and dimension

Dimension:
160 x 200 x 1.5 mm
Resolving gel:
Acrylamide/PDA (9-16% T / 2.6% C)
Stacking gel:
No stacking
Leading buffer:
Tris-HCl (0.375 M) pH 8.8
Trailing buffer:
Tris-glycine-SDS (25 mM-198 mM-0.1% w/v) pH 8.3
Additives:
Sodium thiosulfate (5 mM)
Polymerization agents:
TEMED (0.05%)
APS (0.1%)

The gels were poured until 0.7 cm. from the top of the plates and overlayered with sec-butanol for about two hours. After the removal of the overlay and its replacement with Tris-glycine-SDS (25 mM-198 mM-0.1% w/v) pH 8.3 the gels were left overnight [14].

IPG gel strips transfer

After the equilibration, the IPG gel strips were cut to size. The second dimension gels were overlayered with a solution containing agarose (0.5% w/v) and Tris-glycine-SDS (198 mM-25 mM-0.1% w/v) pH 8.3 heated at about 70o C and the IPG gel strips were immediately loaded through it [8].

Running conditions [14]

Current:
40 mA/gel (constant)
Voltage:
The voltage is non-limiting, but usually requires 100 to 400 V.
Temperature:
8-12o C
Time:
5 hours


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