Nonequilibrium pH gradient

Two-dimensionai gel electrophoresis of histones from interphase and mitotic cells treated with dimethyl sulfate. 2-D gel electrophoresis was carried out with proteins separated by nonequilibrium pH gradient electrophoresis in the first dimension and on 8% polyacrylamide slab gels in the second (6, 7). Separation of histones was enhanced by use of a urea solubilization buffer containing protamines to displace histones from DNA (8). The resulting autoradiograms for histones of both nuclei and chromosomes are in Fig. 3. Incubation of mitotic cells with [32P]NAD before chromosomes were isolated led to prominent labeling of histones H2B and H4 (A, B), and spots are also visible at the positions of HIA and HIB. 32p incorporation is also seen at the positions of histones H2A and H3. hi Fig. 3B, a similar pattern is observed for chromosomal histones from cells treated with DMS. The primary acceptors of isotope are H2B and H4, with radioactivity also present at the positions of HIA, HIB, H2A, and H3. A major change in the relative distribution of 32p between species is clearly not a feature of DMS treatment. 

Fig. 3. Histones, resolved by two-dimensional gel electrophoresis, from cells treated with DMS. [ pjNAD-labeled nuclei and chromosomes were isolated, and the histones were solubilized in protamine-containing buffer (8). Autoradiograms (A) of control chromosomes and (B) from cells treated with 1.0 mM DMS (C) labeled histones of control nuclei and (D) nuclei from DMS-treated (1.0 mM) cells. The isoelectric focusing dimension (horizontal) used nonequilibrium pH gradient electrophoresis. SDS-polyacrylamide slab gels (12.5%) were run in the second dimension (vertical).

High-Resolution Two-Dimensional Gel Electrophoresis of Proteins Isoelectric Focusing and Nonequilibrium pH Gradient Electrophoresis (NEPHGE)... 

Capillary nonequilibrium pH gradient gel electrophoresis in conjunction with mini slab polyacrylamide electrophoresis is a rapid, cost-effective, and easy-to-handle system for the two-dimensional analysis of complex protein mixtures in one day. 

Nonequilibrium pH gel electrophoresis (NEPHGE) uses a pH gradient created by soluble ampholytes. However, the proteins are loaded on the acid end and then electrophoresed. The pH gradient does not really form in a uniform manner and proteins do not focus to a particular location. This method is useful in separating more basic proteins. 

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