Contact Us2D-PAGE
First dimension
The introduction of immobilised pH gradients (IPG) has caused a significant increase in the reproducibility of separations by IEF, and allows the use of narrow pH intervals (Bjellqvist et al. 1982; Gelfi et al. 1986; Gorg et al. 1988).
The cup loading technique with a separated strip rehydration step is a widely used sample application procedure (Barry et al. 2003). Samples can also be applied by in-gel rehydration (Rabilloud et al. 1994) which offers the advantage that sufficient quantities of protein can be loaded from samples with low protein concentrations (Rabilloud et al. 1994). Various protocols for IEF based separations have been described by Görg et al. (Gorg et al. 2000; Gorg et al. 1988).
Using standard wide-range (pH 3–10) IPG strips allows 1000 to 3000 protein spots to be resolved per gel (Hoving et al. 2002). For an increase in the resolution of protein clusters, or for the visualisation of low abundant proteins, narrow pH-range gels, called zoom-gels , are frequently used (Hoving et al. 2002, Wildgruber et al. 2000).
Second dimension
After an equilibration step of the IPG strips, which enhances the protein transfer from the first to the second dimension (Gorg et al. 1988), SDS-PAGE is run in horizontal or vertical systems with the standard buffer system of Laemmli (Laemmli, 1970):
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horizontal second-dimension system : gels are fixed to a plastic support which prevents alterations in gel size during the staining process (Gorg et al. 1980).
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vertical SDS-PAGE system : the systems available are more suitable for high-throughput applications in comparative proteome analysis studies, as they allow multiple gels to be run in parallel, with a corresponding increase in reproducibility (Beranova-Giorgianni, 2003). In addition, they provide the possibility of working with homogenous or gradient-resolving gels (Zhan & Desiderio, 2003).
The use of an acrylamide gradient in the second dimension can increase the resolution of protein separation in a certain Mr range whereas the total range is essentially the same as with linear gels (Scheele, 1975). Protocols for SDS-PAGE are provided by Görg et al. (Gorg et al. 2000; Gorg et al. 1988).