Immobilines

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. 

Righetti PC. (1990) Immobiline pH Gradients Theory and Methodology. Amsterdam Elsevier. 

Figure 3 Two-dimensional gel electrophoresis expression map generated from primary human cells using immobiline strips with a PI range of 3-10 and a molecular size window of 8-120 kDa.

Two-dimensional gel separations were performed on the cell lysates generated. We used Immobiline Dry strips (180 mm, pH 3-10 nonlinear), rehydrated in solubilization solution also containing 10 mM dithiothreitol (DTT), and 0.5% IPG 3-10 buffer. The isoelectrophofocusing (IEF) step was performed at 20°C in a IPGphor. We found it to be important to equilibrate the strips for 10 min using 65 mM DTT, 6 M urea, 30% (w/v) glycerol, 2% (w/v) sodium dodecyl... 

Since temperature has a marked effect on the pK of the carrier ampholytes and immobilines, and on the pi values of proteins, IEF gel runs should be thermostatted to achieve reproducible results. Also, for rapid IEF separations, it is necessary to use higher voltages. This produces a lot of heat which is best removed by running the gel on a cooled plate (10 °C) it is also desirable to even out thermal fluctuations by using stabilised power packs such as those used for DNA sequencing. 

Available from Pharmacia Biotech as Immobiline, these are "derivatives of acrylamide with different dissociation constants. For acidic Immobiline (with pK 3.6 or 4.6), carboxylic acid is chosen as a functional group for the bases, tertiary amino groups are selected. When different pK values of Immobiline are mixed in appropriate proportions, together with acrylamide, bisacrylamide, TEMED, and ammonium persulfate, the buffering groups responsible for ereating the pH gradient become covalently attached to the polyacrylamide base. 

An exciting innovation in lEF has been the development of Immobiline reagents (LKB) for the generation of immobilized pH gradients. 

Immobiline gels are cast in slaTndard vertical cassettes on GelBond PAG supports. After polymerization the gel must be washed to remove unreacted catalysts and unpolymerized Immobilines. The gel swells during this process and must be dried back to its original weight. It is, therefore, much preferable to use a rehydratable gel system, where after washing the gel can be dried, stored and rehydrated for use (see above). 

Elution systems. The recovery from IPG matrices would have to be electrophoretic for two reasons (a) an IPG gel would behave as a veak ion-exchanger (b) even extensively washed gels would still contain short, uncross-linked polyacrylamide-Immobiline chains which would be co-eluted with the protein if the latter v re to be extracted directly from an excised and ground gel zone. There are at least four elution modes, which are given belcw. 

Formulations with Only the Commercially Available Immobilines... 

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