Protein detection, fluorescent silica

FIGURE 10.13 The TLC profiles of labeled peaks isolated from [U- C]ascorbic-acid-modified calf lens protein obtained from Bio-Gel P-2 chromatography. Peaks 2 to 7 were spotted on a preparative silica gel TLC plate and developed with ethanol/ammonia (7 3, v/v). The fluorescence in each lane was detected by irradiation with a Wood s lamp at 360 nm, and the pattern of radioactivity was determined by scanning the plate with AMBIS imaging system. (Reprinted with permission from Cheng, R. et al., Biochim. Biophys. Acta, 1537, 14-26, 2001. Copyright (2001) Elsevier.)... 

Post-column derivatization was employed to detect the separated proteins [657] or amino acids [317,658]. For instance, 2-toluidinonaphthalene-6-sulfonate (TNS) was used to derivatize serum proteins in the post-column format (see Figure 6.34) [657]. With the use of fused silica with substantially lower fluorescence background = 325 nm), a two-fold improvement in S/N for phenylalanine detection was achieved [658],... 

Foote and coworkers [120] developed a microfabricated system with the ability to electrophoretically preconcentrate fluorescently labeled proteins prior to their separation (see Fig. 6). The authors were able to preconcentrate the proteins using a porous silica membrane situated between adjacent microchannels that allowed for the passage of buffer ions, but excluded larger migrating molecules, such as proteins. Preconcentration factors of 600-fold were achieved using this on-chip format followed by an electrophoretic separation of proteins with SDS-PAGE. Using this chip, fluorescently labeled ovalbumin was detected at concentrations as low as 100 fmol by a combination of field-amplified injection and preconcentration at the membrane prior to microchip electrophoresis. 

In CE, the principle detection schemes are spectrometric and electrochemical. Fluorescence is easy to implement (especially off-chip), is extremely sensitive, which is useful since the sample volumes are typically very small, and is well understood. However, some compounds may need to be fluorescently labelled . This can be done prior to, during or after separation. Renzi et al. from Sandia National Laboratories have reported a handheld microanalytical instrument for CE analysis of proteins using laser-induced fluorescence detection ". The fused silica chip is 2 X 2 cm and features on-chip sample introduction, inlet port filters and a 10 cm separation column. Nanomolar concentrations of fluores-camine-labelled proteins were detected. 

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