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Imidazole-zinc reverse stain utilizing imidazole and zinc ions for protein visualization on electrophoresis gels was originally introduced in the 1990s (1,2,3). This method is based on the selective precipitation of imidazolate-zinc complex in the gel (3), except zones where proteins or other macromolecules, such as DNA (4,5,6,7) or lipopolysaccharide, are present (4,5,8,9). There are several advantages of using imidazole-zinc reverse stain in current proteomic research. First, imidazole-zinc reverse stain is highly sensitive. For protein gels, zinc reverse stain has been demonstrated to provide an equal or better staining sensitivity than Sypro Ruby stain or some silver stains (4,5). Protein, as low as 1 ng, may be detected in the gel by the reverse stain. Second, performing imidazole-zinc reverse stain is exceedingly simple as compared with silver stain, which requires tedious preparation of fresh staining solutions. Imidazole-zinc reverse stain uses only two staining solutions, which can be easily diluted from stock solutions. Third, it is remarkably fast, generally taking less than 20 min to complete (4,5,10). Finally, imidazole-zinc reverse stain is fully compatible to down-stream applications, such as mass spectrometry (MS) (4,11,12,13,14), Edman sequencing (1,2,3), electroelution (1,2,4,6), and membrane blotting techniques (4,5). Although the dynamic range of staining for imidazole-zinc reverse stain might not be as satisfactory as for Sypro Rube stain, nowadays laboratories that are not equipped with synchronized spot pickers on their fluorescent scanners sometimes use it to restain the Sypro Ruby stained two-dimensional electrophoresis (2-DE) gels, then manually pick the interested protein spots for identification by MS.
The imidazole-zinc reverse stained gel delivers an image with transparent bands, spots, and a white background. When the gel is placed above a dark background, the negative gel image can be converted to a positive image with black bands, spots, and a white background. However, observations of gel images seen by the eyes are generally worse than those acquired by scanners or charge-coupled device (CCD) cameras. When documenting images by applying those instruments, it is possible to adjust the parameters, such as brightness, contrast, or γ ratio to obtain high-contrast gel images. It is impossible to perform such image adjustments with the human eye; therefore the observed images are not as discernible as those obtained by machines, which, consequently, results in the potential of incorrect picking of minor protein spots, leading to the misjudgment of protein identities by subsequent analysis of MS. Due to the inferior observation result of imidazole-zinc reverse stained gel using the conventional setup, its utilization in current proteomic research is limited, especially when minor proteins are the targets.
In this article, we present a setup to enhance the image quality of observed imidazole-zinc reverse stained gels. Compared with the original setup that utilizes a dark background for observation (4, 5), our new setup utilizes a backlit light plate to illuminate the gel. The light plate assembly is shown in Figure 1A. The transparent plate on top of the light plate can be made of glass or acrylic for light transmission. One or two linear cold cathode fluorescent lamps (CCFLs) are placed sideways of the transparent plate as the light source. A black background is placed underneath the transparent plate for better contrasting (such light plate, which is often used in restaurants or stores for advertisement purposes). When a fluorescent water-based chalk pen is used, the drawn objects will glow as the light is turned on. With illumination, a significant increase in contrast between the drawn objects and the black background appears.
As compared to the original setup, the light plate setup was found to considerably enhance the quality of the observed image of imidazole-zinc reverse stained gel (see Figure 1, C and D). In addition, this setup allows researchers to see protein bands with lower abundance in the reverse stained gel. For instance, with the aid of the light plate, when a commercial protein standard mixture (576 µg/vial low molecular weight protein standard; GE Healthcare, Piscataway, NJ, USA) with 2-fold serial dilution (starting from 8 µg to 15.6 ng total proteins) was separated by a 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, the lowest protein amount from carbonic anhydrase (83 µg/vial) that could be seen was about 2.25 ng (15.6×83/576) in the reverse stained gel (Figure 1D, red arrow). However, using only a black background, 4.5 ng carbonic anhydrase (31.2×83/576) was merely detectable (Figure 1C, red arrow). The quantitative analysis of above gel images was performed by using image analysis software (TotalLab 100; Nonlinear Dynamics, Durham, NC, USA). The overall volumes (expressed as pixels) of protein bands from the gel image in Figure 1D (Figure 1F) were approximately 2- to 4-fold higher than the ones in Figure 1C (Figure 1E). It was also found that using this novel method, the quality of the observed gel image was almost identical to the scanned image by machines (Figure 1B, documented by a flatbed scanner). The light plate method, therefore, should enable researchers to see minor protein spots from the reverse stained 2-DE gels. This method, in addition, was found to improve the observed images for the reverse stained DNA gels (see Supplementary Figure S1 available online at www.BioTechniques.com).
The possible mechanism for the light plate setup was proposed as following. According to Snell's Law, between two transparent materials with different refractive index (n1 and n2), the traveling path of light from material 1 to material 2 should follow:
When the path of incident light is vertical to the interface (incident angle = 0°), the majority of light emitting from material 1 will pass through material 2 and the minority will be reflected back (Figure 2A, path a). When the incident angle is larger than 0°, refractive light follows Snell's law (Figure 2A, path b). Under the circumstance for n1 > n2, total reflection will occur when the incident angle θ1 is larger than critical angle θc (Figure 2A, paths c and d). The critical angle θc is calculated as follows:
For instance, the critical angle θc between glass and air is approximately 41.81°. This means that light emitting from glass to air with incident angle θ1 > 41.81° will be totally reflected back into glass. In the case of the glass-made light plate, since the linear light source is located sidewise of the glass plate, almost all light is reflected back and travels along within the glass plate (Figure 2B, path 2). The path of refractive light should be similar to those in the application of optical fiber. Therefore, even when the sidelight is turned on, the light plate still appears dim since a black background is placed underneath the glass plate (Figure 1A). However, if any nontransmittable substance, such as white powder or dust, is on or in the glass plate, the incident light will glow at those areas. Consequently, the commercial light plate delivers exceptional contrast between the black background and the drawing characters.
The refractive index for polyacrylamide gel has been estimated as 1.47; however, it is intriguing since in previous studies the concentration of the gels is not specified (15). Nevertheless, since the concentration of most polyacrylamide gel used in protein electrophoresis is larger than 10%, it is reasonable to assume that the refractive index of 10% polyacrylamide gel is between 1.33 (water) and 1.47. When such gel with n=1.47 is placed above a glass plate with a side light turned on, most light will enter the gel (Figure 2B, path 3) unless the incident angle is >78.52° (the critical angle θc between glass and polyacrylamide, Figure 2B, path 4). Consequently, when a zinc reverse stained gel is placed above the glass plate, most light travels into the gel and glows at the area where white zinc-imidazolate complex exists. The contrast between protein black bands and white background is enhanced (Figure 1D).
This light plate setup should significantly increase the applicability of imidazole-zinc reverse stain in current proteomic research.
The authors would like to acknowledge Drs. Lila R. Castellanos-Serra and Eugenio Hardy (Center for Genetic Engineering and Biotechnology) for valuable suggestions during manuscript preparation. Part of this work was supported by the research grant (93-2314- B-030-001) from National Science Council (ROC) and the Foundation of Research in Catholic Fu-Jen University (9991A15, 10953104983-2).
The authors declare no competing interests.
