11
Figure 6: IEF separation process of 5 pI markers monitored in an iCIEF instrument. Frame order from left to right and from top to bottom. (frames are courtesy of Martin Donker of Isogen Lifescience, Netherlands)
to completion with the zones at the low pI nearly and on the high pI side already focussed. In frame 6 after 8 minutes, the focusing has completed.
In a practical example, the separation of haemoglobin isoforms is shown in Figure 7.
Method development for iCIEF
Figure 7: Separation of haemoglobin A, F, S, C isoforms. Lower trace 100 µm i.d. separation ca- pillary, top trace 200 µm i.d. separation capillary. Sample concentration 1 mg/mL. (Figure courtesy of Tiemin Huang, Advanced Electrophoresis Solutions)
Given the absence of a mobilisation step and the short length of the separation capillary, the analysis cycle time will be short compared with a conventional CIEF method.
The first step is the sample preparation containing the proteins to be analysed (approx. concentration 0.1-0.2 mg/ml), 4% ampholyte with pI’s ranging from pH 3-10, and additives as required. The evolving
separation is monitored continuously as illustrated in Figure 8.
This figure illustrates the benefit of continuously monitoring the development of the separation. In CIEF, only the current is taken as an indicator for completion of the focusing. But since the focussing process cannot be observed, it is arbitrary to set the proper focussing time. As shown by iCIEF, at 2.5 minutes, the current is very low and does not decrease as rapidly as earlier, but the focussing is far from complete.
With the data from the initial experiment reviewed, a narrow range pI ampholytes fitting the focussed region allows for the separation of the sample components in the same length of the separation capillary with higher resolution as illustrated in Figure 9.
Comparison CIEF and iCIEF
A comparison of both techniques is shown in Table 1 focusing on the salient features of both methods. Capillary Electrophoresis equipment offers an open platform for the execution of a diverse range of electrophoretic separations such as Capillary Zone Electrophoresis, Micellar ElectroKinetic CE, Capillary Gel Electrophoresis (esp. SDS- PAGE), Capillary Electro-Chromatography, Capillary Isotachophoresis, and CIEF. However, from a pragmatic perspective the equipment is typically allocated to a specific analysis method and so this flexibility is not always realised. Therefore, this benefit diminishes compared to using a dedicated iCIEF instrument.
Figure 8: Sample: Hemoglobin A, F, S, C isoforms, 8% Pharmalyte 3-10, field 600 V/cm (Figure courtesy of Tiemin Huang, Advanced Electrophoresis Solutions)
iCIEF analysis cycle time (including the time needed for filling of the separation
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