As shown in Fig. 4, each cm of gel contains proteins having isoelectric point differing in one unit. This may accommodate more protein per slice and effective purification may not be achieved in a protein mixture having many proteins.
By carefully choosing range of ampholytes better separation may be achieved. For example if we know the isolectric point of desired protein, range of ampholytes can be narrowed. If we consider that the desired protein's isoelectric point is 5.0, we may use ampholytes of range 4-7 (3 unit pH difference) in 10 cm gel. This will result in 1cm slice accommodating proteins differing in 0.3unit (10cm/3unit pH difference) difference in isoelectric point.
Other less common method for generating pH gradient
Some times immobilines are also used to generate pH gradient. Immobilines are derivatives of acrylamide. It could be acidic or basic and polymerized (as we have seen in case of acrylamide). Solutions of acidic and basic immobilines are used to generate pH gradient as shown in Fig. 5. Once pH gradient is generated, the gel can be used for isoelectric focusing.
Figure 5: Preparation of pH gradient using immobilines
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