Nonlinear Extended pH Gradients

IPG formulations have been given only in terms of rigorously linear pH gradients. While this has been the only solution adopted so far, it might not be the optimal one in some cases. Altering the pH slope in some portions of the gradient might be required in those pH regions overcrowded with proteins. The reasons for resorting to non-linear pH gradients are given in the histogram of Figure 7. With the relative abundance of different species it is clear that an optimally resolving pH gradient should have a gentler slope in the acidic portion, and a steeper course in the alkaline region. Such a general course has been calculated by assigning to each 0.5 pH unit interval in the pH 3.5-10 region a slope inversely proportional to the relative abundance of proteins in that interval. The ideal (dotted) curve in Figure 7 was obtained by such a procedure. What is also important here is the establishment of a new principle in IPG technology, namely that the pH and density gradients stabilizing it need not be co-linear. The possibility exists of modulating the former by locally flattening of pH

Figure 8 IEF of conalbumin in an IPG pH 4.5-6.5 gradient. Gel: 5%T, 3%C Polyacrylamide, equilibrated in 10% glycerol. All samples were applied in round basins punched through the gel thickness at the cathodic side as 20 ^L droplet (20-500 ^g protein). Staining with Coomassie Blue R-250 in ethanol/aceticacid in presence of copper sulfate. Notice that, although the gel thickness is only 0.5 mm, there is no overloading effect in such a wide interval of protein concentration (from Righetti PG and Ek K, unpublished observations).

Figure 8 IEF of conalbumin in an IPG pH 4.5-6.5 gradient. Gel: 5%T, 3%C Polyacrylamide, equilibrated in 10% glycerol. All samples were applied in round basins punched through the gel thickness at the cathodic side as 20 ^L droplet (20-500 ^g protein). Staining with Coomassie Blue R-250 in ethanol/aceticacid in presence of copper sulfate. Notice that, although the gel thickness is only 0.5 mm, there is no overloading effect in such a wide interval of protein concentration (from Righetti PG and Ek K, unpublished observations).

IPG pH 10-12 Histories

Figure 9 Focusing of histones in an IPG pH 10-12 nonlinear interval. Gel: 6% T, 4% C polyacrylamide matrix, containing an IPG 10-12 gradient, reswollen in 7 M urea, 1.5% Nonidet P-40 and 0.5% Ampholine pH 9-11. The gel was run at 10°C under a layer of light paraffin oil at 500 V for the first hour, followed by increasing voltage gradients, after sample penetration, up to 1300 V for a total of 4 h. The samples (2 mg mL~1, 50 |L seeded) were loaded in plastic well at the anodic gel surface. Staining with Coomassie Brilliant Blue R-250 in Cu2 + . Histone samples (from left). (1) VII-S (Lys-rich); (2) VI-S; (3) II-AS and (4) VIII-S (Arg-rich, subgroup F), from calf thymus. The p/10.6 marker (cytochrome C) is in track 5 on the right side (from Bossi etal., 1994, by permission of Elsevier Science Publishers).

Figure 9 Focusing of histones in an IPG pH 10-12 nonlinear interval. Gel: 6% T, 4% C polyacrylamide matrix, containing an IPG 10-12 gradient, reswollen in 7 M urea, 1.5% Nonidet P-40 and 0.5% Ampholine pH 9-11. The gel was run at 10°C under a layer of light paraffin oil at 500 V for the first hour, followed by increasing voltage gradients, after sample penetration, up to 1300 V for a total of 4 h. The samples (2 mg mL~1, 50 |L seeded) were loaded in plastic well at the anodic gel surface. Staining with Coomassie Brilliant Blue R-250 in Cu2 + . Histone samples (from left). (1) VII-S (Lys-rich); (2) VI-S; (3) II-AS and (4) VIII-S (Arg-rich, subgroup F), from calf thymus. The p/10.6 marker (cytochrome C) is in track 5 on the right side (from Bossi etal., 1994, by permission of Elsevier Science Publishers).

gradients for increased resolution, while leaving unaltered the latter.

Although only one example of a nonlinear extended pH gradient is given here, clearly the possibility exists of modulating in the same fashion any narrower pH interval.

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