Matrix immobiline

Problems associated with gel-to-gel variabiUty have been rectified with the advancement of ampholyte mixtures. One commonly used mixture of ampholytes, called Immobilines, is an improved ampholyte mixture that produces no gradient drift or unequal pH gradient (26) and can be used in a gel matrix reproducibly from one day to the next. 

Fig. 12. Polymerization efficiency of the seven Immobiline species as a function of temperature. The percentage incorporation into the matrix is putative, as it is based on the ratio between initial and final absorbancies at 285 nm (disappearance of double bonds). The best convergence (similar reactivity ratios) is only obtained at 50°C. Note that the critical parameter is not so much a 100% incorporation efficiency, but the achievement of a ratio of incorporation between the buffering ion and the titrant as close as possible to unity. (From Righetti et al., 1984a. Reproduced with permission of the publisher.)...

Fig. 17. The complete device for mixing an Immobiline gel gradient. (l)-(3) Computer, floppy disk station and matrix printer (4) the four burettes, A to /) (5) the mixing chamber on a (6) magnetic stirrer (7) a polymerization stand with two separation cells to be filled from the bottom. (From Altland"and Altland, 1984. Reproduced with permission of the publisher.)...

For small-scale protein loads in IPGs, an interesting method was described by Bartels and Bock (1984) who recovered the protein of interest focused in the Immobiline gel directly into gel filtration media this was performed by collecting the protein of choice into a layer of Sephadex G-200, inserted into a channel cut into the IPG matrix. 

Figure 4 Hypothetical structure of an Immobiline gel and mechanism of the focusing process. The acrylamide acid and basic groups are shown grafted onto the polyacrylamide matrix. Two proteins are shown migrating in the gel at the times f = 0, at f = 1 and finally at the steady state, where they reach their respective pi values (pli and pla) as points of zero net charge.

This is a modification of lEF in which the pH gradient is not created by the electric field but is preconstituted by covalently grafting on to a polyacrylamide matrix a series of buffering and titrant acrylamide derivatives (known commercially as immobilines). 

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