Narrow range, IPG

Figure 2. A 2D proteome map of L. major Friedlin. Whole cells were extracted with a lysis buffer containiug urea, thiourea, and CHAPS. Samples were run using a variety of narrow-range IPG strips (4.0-5.0, 4.5-5.5, 5.0-6.0, 5.5-6.7, and 6-11 Amersham), gels were silver-stained, and the images were merged using PDQuest software (BioRad).

Figure 4. Comparative proteome analysis of L. donovani infantum axenic promastigotes and amastigotes. Proteins from whole cells were extracted as described in Figure 2, run on narrow-range IPG strips, and stained with SYPRO Ruby (Molecrdar Probes) to allow quantitative comparisons. Representative gels of each stage are shown. The expanded region contains a number of differentially-expressed spots. Spots marked with orange arrows appear to be stage-specific, while spots marked with blue arrows are differentially expressed.

Alkaline proteins with similar pi values (e.g., ribosomal and nuclear proteins with pis in the range of 10.5-11.8) can be differentiated by narrow range (pH 10-12 or 9-12) IPG gels. Substitution of DMAA (N,N-dimethylacrylamide) and addition of propan-2-ol to the rehydration solution provide high reproducibility and suppresses the reverse electroendosmotic flow. To obtain better resolution for this type of... 

It had first to be demonstrated that proteins would quantitatively elute from IPG strips and run in SDS gel electrophoresis according to their molecular mass and irrespective of their focusing position along the pH gradient. The absence of interference from ionic interaction or adsorption effects by the charged IPG matrix was in fact demonstrated by others (19, 40) for narrow and by us (21) for wide pH ranges. 

For the first-dimension separation, a protein sample containing an appropriate cocktail of carrier ampholyte is applied to a standard immobilized pH gradient gel (IPG) strip and left overnight to rehydrate the strip. These strips are available in several narrow pH ranges for improved separation and are specially well suited for FFE-separated subfractions. Application of the appropriate electrical potential to the rehydrated strip results in separation of the proteins according to their intrinsic charge. 

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