Fluorescent Detection Methods

Many of the problems inherent in the quantification of gel-separated proteins visualized by silver staining can be overcome using detection methods based on the use of fluorescent compounds. This group of methods is highly sensitive and generally exhibits excellent linearity and a high dynamic range, making it possible to achieve good quantitative analysis, particularly if a suitable imaging device is used.

Two approaches can be used, the first being to couple the proteins with a fluorescent-labelled compound prior to electrophoresis. Examples of such compounds are: dansyl chloride; fluorescamine (4-phenyl-[furan-2(3H)-1-phthalan]-3,3'-dione);

0-pththaldialdehyde (OPA) # a thiol; and MDPF (2-methoxy-2,4-diphenyl-3(2H)-furanone). The last reagent has a reported sensitivity of 1 ng protein per band and is linear over the range 1-500 ng protein per band.

The main disadvantage of pre-electrophoretic staining procedures is that they can cause proteincharge modifications, for example fluorescamine converts an amino group to a carboxyl group when it reacts with proteins. Such modifications usually do not compromise SDS-PAGE unless a large number of additional charged groups are introduced into the protein. However, they result in altered mobility during other forms of electrophoresis, giving rise to altered separations by native PAGE, IEF and two-dimensional electrophoresis. Recently, compounds that react with cysteine or lysine residues have been described and used successfully for two-dimensional electrophoresis separations. The cys-teine-reactive reagent monobromobimane has been used to label proteins prior to analysis by two-dimensional electrophoresis. Using a cooled CCD camera to measure fluorescence, the limit of detection was found to be 1 pg protein per spot.

In an alternative approach, two amine-reactive dyes (propyl Cy3 and methyl Cy5) have been synthesized and used to label Escherichia coli proteins prior to electrophoresis. These cyanine dyes have an inherent positive charge, which preserves the overall charge of the proteins after dye coupling. The two dyes have sufficiently different fluorescence spectra that they can be distinguished when they are present together. This allowed two different protein samples, each labelled with one of the dyes, to be mixed together and subjected to two-dimensional electrophor-esis on the same gel. This method, which has been termed 'difference gel electrophoresis (DIGE)', has great potential for improving the efficiency of detection of differences in two-dimensional electrophoresis protein profiles between different samples.

For two-dimensional electrophoresis, one approach to overcoming the problems associated with charge modification during the IEF dimension is to label the proteins while present in the first dimension gel after IEF, prior to the second dimension separation by SDS-PAGE. Two fluorescent labels that have been used in this way are MDPF and a fluorescent maleimide derivative.

The alternative approach, which also overcomes the problem of protein-charge modifications, is to label the proteins with fluorescent molecules such as

1-aniline-8-naphthalenesulfonate (ANS) and OPA after the electrophoretic separation has been completed. However, these two methods suffer the disadvantage of relative insensitivity. Recently, two post-

electrophoretic fluorescence staining reagents, SYPRO orange and red (Molecular Probes, Eugene, Oregon, USA), have been described. These stains have a very high sensitivity (1-2 ng protein per band) and excellent linearity with a high dynamic range. Using a fluorescence imaging device, the SYPRO dyes have been shown to be linear over three orders of magnitude in protein quantity. The other advantage of this method is that staining can be achieved in only 30 min, compared with staining with silver and CBB R-250 which can take from 2 h to overnight. Gels can be stained without fixation so that they can be subjected to subsequent Western blotting procedures. However, staining with these reagents requires that the proteins are complexed with SDS, so that if the gels are fixed prior to staining or electrophoresis is carried out in the absence of this detergent, then the gels must be incubated in a solution of SDS prior to staining. An SDS-PAGE gel separation visualized using SYPRO red is shown in Figure 3.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

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