Protein Protocols Protein Protocols

172 Gianazza
Table 1
pK Values of Immobilines
Water solution, PAA gel, 8 M urea
Chemicals 25°C a 10°C a in PAA gel, 10°C b
Immobiline pK 3.6 3.58 3.57 4.47
Immobiline pK 4.6 4.61 4.51 5.31
Immobiline pK 6.2 6.23 6.21 6.71
Immobiline pK 7.0 6.96 7.06 7.53
Immobiline pK 8.5 8.52 8.50 8.87
Immobiline pK 9.3 9.27 9.59 9.94
a From LKB Application Note 324.
b From ref. 42.
6. 1 M Acetic acid: Dilute 57.5 mL of glacial acetic acid to 1 L.
7. Dimethyldichlorosilane solution: 2% v/v solution of dimethyldichlorosilane in 1,1,1,trichloroethane.
Both chemicals are available from Merck (Darmstadt, Germany), or a
ready-made solution is marketed by Pharmacia as RepelSilane.
8. Glycerol washing solution: 1% v/v glycerol.
3. Method
3.1. Selecting a Formulation
The formulations for narrow to wide-range IPGs have been reported in a series of
papers (12–15), and the recipes for casting the gradients collected (2). Most of the
formulations use the acrylamido buffers marketed as Immobiline from Pharmacia,
since the pK distribution in their set is even and broad enough for all applications in the
4.0–10.0 pH region. For specific purposes—increased hydrophilicity, narrow ranges,
pH extremes—other chemicals are available as acrylamido derivatives (16) or have
been custom-synthesized (17,18). These chemicals are available from Fluka.
Computer programs for pH gradient modeling, with routines optimizing the concentrations
of the acrylamido buffers required in the two limiting solutions in order to give
the expected pH course, have been described in the literature (19–21). One of these
programs is available from Pharmacia (Doctor pH). For most practical applications,
a wide pH gradient is required for 2-D mapping in order to resolve all components of a
complex biological sample. A statistical examination of the pI distribution across all
characterized proteins as well as the typical maps of most biological specimens show a
prevalence of acidic to mildly alkaline values over basic proteins. A nonlinear pH
course is then expected to give improved resolution of most proteins in a complex
sample. An exponential gradient whose slope increases from pH 6.0 to 7.0 and to 8.0
optimally resolves most samples run in IPGs: one such recipe (14) is given in Table 2
(I), whereas the gradient is plotted in Fig. 2.
The narrow ranges in Table 2 (II–IV) have been interpolated from the sigmoidal gradient
above, and provide a better resolution of the acidic, neutral, and basic protein components
(Fig. 3) while maintaining the same slope ratios among the different pH regions.
Alternatively, resolution may be improved by the parallel use of two linear, partly overlapping
gradients, namely 4–7 and 6–10 (12) (Table 3).