Fluorescence during electrophoresis experiments

Many workers have observed fluorescent areas or fractions during electrophoresis experiments (e.g., Waldron and Mortensen, 1961), and this behavior is usually associated with the more mobile materials. It is not clear whether the fluorescence is due to the presence of closely associated non-humic components, or is an innate characteristic of particular fractions of humic substances. It is possible that all fractions do in fact fluoresce, but that this fluorescence is masked by the intense light absorption of the gray-brown components in fractions which do not emit measurable fluorescence. 

We have developed two-dimensional CE systems for the characterization of proteins and biogenic amines. The use of this technology for chemical cytometry is similar to the use of onedimensional electrophoresis a cell is aspirated into the column, lysed, and its components labeled with FQ. For two-dimensional electrophoresis, components are separated based on CSE in the first-dimension capillary. Fractions are then transferred across an interface to a second capillary, where they undergo additional separation based on micellar electrokinetic chromatography (MECC) before detection by fluorescence. The voltage drop across the first capillary is set to zero during the second dimension separation, holding components stationary. In a typical experiment, 300 fractions are transferred between capillaries under computer control. 

Figure 14.13 shows the sample protein assay results of using a commericial 100-kDa microdialysis probe (PES dialysis membrane, an effective length 10 mm, 0.4 mm OD) to monitor bovine intervetebral disk (IVD) cultore (Li et al., 2006). The IVD is cultured up to 7 days, and the probe is operated by either push, puU, or push-and-puU modes. Apart from glucose and lactate, the probe picked up at least three proteins with a molecular weight around 40 kDa. Three peaks of proteins (PI, P2, and P3) were evident on fast protein liquid chromatography (FPLC) outcome during the 7-day culture period. Three bands were also evident using 12% SDS PAGE electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis) with silver stain (see Fig. 14.13). In these experiments, the relative recovery is easily determined using the developed in situ calibration using 40-kDa fluoresecent dextran as the internal standard. In the 7-day experiments, the relative loss of fluorescent dextran (40 kDa) stablized at 8 -10%, which is equivalent to the protein transmission across the membrane (Li et al., 2006).

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