New optical filter for Eagle Eye II still video system
Michelle Cayouette Jane Moores
Stratagene Cloning Systems, Inc.
Stratagene currently provides an optical bandpass filter with all Eagle Eye II still video systems that is optimized for visualizing ethidium bromide fluorescence. However, a single filter specifically designed for ethidium bromide may be inadequate for use with other fluorophores or stains. In this report, we describe an optical filter designed for use with protein gels stained with Coomassie brilliant blue dye. The Coomassie blue bandpass filter enhances contrast and sensitivity for stained protein gels when compared to the ethidium bromide-optimized filter. Sensitivity was further increased when Coomassie blue-stained gels were imaged using a combination of the Coomassie blue-optimized filter and overhead illumination with the gel resting on top of a piece of white acrylonitrile butadiene styrene (ABS) plastic. The addition of the Coomassie blue bandpass filter expands the line of optical filters to the Eagle Eye II system optimized for specific applications.
Optical filters are placed in front of a camera lens to isolate or enhance the contrast between specific wavelengths of light. Stratagenes line of optical filters for the Eagle Eye II still video system currently consists of two filters. The bandpass filter, provided as part of the Eagle Eye II still video system, is designed to maximally overlap the emission spectrum of ethidium bromide, a fluorophore that emits orange light upon excitation with ultraviolet light. This ethidium bromide-optimized filter preferentially allows orange fluorescent light to pass to the charge-coupled camera (CCD) camera, excluding all non-orange wavelengths of the spectrum. The other filter for the Eagle Eye II still video system is the Evergreen Filter, an optical bandpass filter designed for SYBR Green nucleic acid stain and FLASH CAT applications.1 The Evergreen Filter maximally overlaps the emission spectra of both of these green fluorophores, assuring optimal sensitivity and signal capture efficiency.
In this report, we describe an optical filter specifically designed for use with Coomassie brilliant blue-stained protein gels. Unlike ethidium bromide, SYBR Green and BODIPY stains, Coomassie brilliant blue is not a fluorescent dye. Proteins stained with Coomassie blue appear blue in color because the stain absorbs non-blue wavelengths of visible light while reflecting blue light. With reflected light, contrast control filters can be used to increase the contrast between an object and its background.2 In this way, the apparent brightness of a colored object can be increased by photographing it through a filter of a complementary color. We have designed a contrast control filter with a narrow bandpass for visualizing Coomassie blue-stained protein gels. As are the other Eagle Eye optical filters, the Coomassie blue bandpass filter is coated to ensure high image quality.
In order to compare the performance of optical filters when imaging Coomassie blue-stained protein gels, various protein preparations were fractionated through a 4-20% SDS-PAGE mini-gel and stained with Coomassie brilliant blue dye using standard conditions.3 The gel was positioned on top of a white-light box and imaged using white-light transillumination. The Eagle Eye II still video system was fitted with either the ethidium bromide optical filter or the Coomassie blue bandpass filter. Images were also taken using each filter with overhead white-light illumination while the gel was resting on top of a pie ce of white ABS plastic. This technique of capturing an image takes advantage of the overhead white-light illumination provided inside the cabinet of the Eagle Eye II still video system and can be used by researchers who either do not have a white-light box or do not want to replace the UV transilluminator with the white-light box prior to imaging a gel stained with Coomassie blue dye.
Figure 1A illustrates the comparison of optical filters. Compared to the standard ethidium bromide-optimized optical filter (Figure 1A), the Coomassie blue bandpass filter enhances contrast and improves sensitivity in images of Coomassie blue-stained protein gels (Figure 1B). Sensitivity was further increased when the gel was placed on top of a piece of white ABS plastic, and the Coomassie blue-optimized filter was used in conjunction with overhead white-light illumination (Figure 1C). However, this method also has the potential of increasing nonspecific background. When the ethidium bromide filter was used in conjunction with overhead white-light illumination and a piece of white plastic, sensitivity was increased when compared to the ethidium bromide filter used alone (data not shown). However, the combination of the Coomassie blue bandpass filter, overhead white-light illumination and a piece of white ABS plastic gave the most sensitive results.
The Coomassie blue bandpass filter expands the line of optical filters for the Eagle Eye II sti ll video system to include a contrast control filter for specific reflective stains. When compared to the standard ethidium bromide-optimized filter, this newly developed optical filter designed for use with blue reflective stains improves contrast and sensitivity for imaging Coomassie blue-stained gels.
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Eastman Kodak Company. (1990) Handbook of KODAK Photographic Filters, p. 20. Rochester, New York.
Maniatis, et al. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.