Fluorescence Gel Imaging Applications of the UVP Gelstudio

A number of DNA and protein detection reagents are available to researchers, which vary in safety, sensitivity, and suitability for downstream applications. Ethidium bromide is chief among DNA binding dyes, with detection limits as low as 0.5 ng¹.

Ethidium bromide, or homidium as it is known in the veterinary sciences, was utilized to effectively treat trypanosomiasis in cattle as early as the 1950s, for which the mechanism of action was unknown until recently².

The dye interferes with trypanosome DNA replication², as any molecular biologist would have expected. This underscores the mutagenic safety concerns with this and other DNA-binding dye technologies.

Ethidium bromide-agarose gel electrophoresis is still the most common fluorescence-based DNA detection technique since its conception³, while ethidium bromide has fallen out of favor in the treatment of cattle for the aforementioned risks.

Yet, a number of alternative fluorescence-based DNA dyes are available to investigators (See Figure 1 and Table 1). Colorimetric dyes are still the most common for protein and Coomassie Brilliant Blue (CBB) is arguably the most common post-electrophoresis protein-dye.

Figure 1. Popular fluorescent dyes for DNA and protein detection within the visible spectrum. SYBR Green, SYBR Gold, SYBR Safe, GelGreen, GelRed, and ethidium bromide (EtBr) are considered the most popular DNA binding dyes for in-gel fluorescence detection. SYPRO Orange, SYPRO Ruby, SYPRO Red, and SYPRO Ruby are the most popular protein binding dyes for in-gel fluorescence detection. SYBR Safe, GelRed, and GelGreen are considered safer alternatives to EtBr. SYBR Gold and SYPRO Ruby are considered the most sensitive dyes for DNA and protein detection, respectively.

Multiple versions of the original protocol exist with reported detection limits which are as low as 3 ng⁴. Alternatively, silver salt staining can increase the detection limits of protein as low as 0.1 ng. Generally, in routine protocols, silver staining acquires 100x-1000x more sensitivity than CBB⁵.

Both techniques still either need laborious and time-consuming staining protocols or large volumes of toxic reagents. Fortunately, there are a number of bright fluorescence-based protein dyes available to investigators which have vastly streamlined in-gel protein detection (Table 1).

Analytik Jena has designed their instruments to accommodate both old and new technologies. Crucially, all of their instruments can image gels stained with the most popular fluorescent dyes on the market (Table 1).

Table 1. DNA and Protein Binding Dye Specifications and Filter Requirements.

These include safer DNA dyes like GelGreen, GelRed, and SYBR safe, in addition to high sensitivity SYPRO Ruby and SYBR Gold for protein and DNA detection, respectively. The diversity of dyes that can be captured with the imagers from Analytik Jena are demonstrated below.

Fluorescence imaging with Analytik Jena’s UVP gelStudio

In order to capture the finest sample details, the UVP GelStudio Series imagers are equipped with a high-performance 5 megapixel camera.

With RGB LED epi-illumination, a UV transilluminator, an easily accessible filter wheel, and an extensive library of emission filters, the UVP GelStudio is able to image any fluorescent dye technology. The flexibility of the UVP GelStudio by imaging is demonstrated below with multiple DNA and protein fluorescent dye technologies.

Methods DNA gels and protein gels

DNA samples were separated on 2 % agarose at 120 volts for at least 45 minutes in 1 X TAE buffer. Biotium 1KB ladder was diluted 1:2 and 20 ul was loaded per well. Before loading DNA, all dyes were homogenized with agarose.

No post-staining or destaining was carried out. The dye concentrations utilized were per the manufacturer’s recommendations. Protein samples were boiled at 85 ^oC for 5 minutes in Laemlli Buffer and separated on pre-cast NuPage 4-12 % Bis-Tris protein gels.

For SYPRO Ruby detection, samples were diluted 1:10 (from 40 ug to 40 ng) and 1:2 (from 20 ug to 156 ng) for SYPRO Red. SYPRO dyes were employed as per the manufacturer’s recommendations. The rapid protocol was used for SYPRO Ruby.

Visionworks software image capture

Images were captured using the VisionWorks ver. 9.0 software package following the steps below (See Figure 2).

1. Navigate to the Devices menu and then the Filters submenu to choose the appropriate emission filter for the application. Up to five preset filter options can be programmed.
2. Select excitation light in the Lights pane.
3. Select Manual (M) and toggle the arrows to adjust the exposure time in the Camera sub-menu. It is recommended to begin with a 200 ms exposure for most gel applications.
4. Select Live View to begin preview. During Live View, navigate to the Lens tab and adjust the Brightness and Focus to your desired level. Once you are satisfied, select Capture in the Camera sub-menu.
5. Navigate to the Image tab and select Histogram (Hist) after capturing an image. Auto is recommended. Alternatively, Manual (M) can be selected, and the black, white, and gray levels adjusted to your liking.

The histogram briefly indicates the pixel counts in regions of the white part of the spectrum (right), the black part of the spectrum (left), and in the gray part of the spectrum (middle or everything in between).

As with the majority gel applications, since the gel background is black with fluorescent signal, most of the signal will be in the black regions. This will be indicated by a right skewed histogram (Figure 2, Histogram Adjustment).

Moving the black slider to the right will drop off background in the image and so will sliding the gray slider to the left. Moving the white slider to the left will bring up the white levels (e.g. DNA bands) in addition to any background signal.

Crucially, none of these modifications alter the metadata of the image, so consider this a tool to help you locate and demarcate bands for subsequent analysis or for simple image beautification.

Figure 2a. Capturing a fluorescent gel image with VisionWorks software ver. 9.0. In the DNA or Protein application, navigate to Devices menu and the Filters sub-menu to select the emission filter for your application. Navigate to the Lights sub-menu and select the excitation light source. Both epi and trans illumination are available (trans lighting option shown above). Next, navigate to the Camera sub-menu, select manual capture (M), and adjust the exposure time as determined empirically (continued in Figure 2b).

Figure 2b. Starting with 200 ms is recommend. Next, select Live View. During Live View, adjust the Focus and Brightness settings until you can clearly see bands. You may need to increase your exposure time to improve visualization. Once the preview image is optimized, select Capture. You can begin post-processing. You want to adjust the histogram to improve the aesthetics of the image. Moving the black slider increases the black levels. Moving the white slider left, increases the white levels. Adjusting the gray slider will change the midpoint of the scale. The gray slider is especially useful if you use a dye with high background fluorescence. Lastly, navigate to the Devices sub-menu Pcolor to apply a pseudocolor to the image.

Results and conclusion

Some of the most popular dyes employed for fluorescence-based DNA and protein detection are readily captured by the UVP GelStudio imager. In Figure 3, the flexibility of the GelStudio imager is demonstrated by using multiple fluorescent dyes covering the visible spectrum from green to red.

Figure 3. Fluorescent DNA and Protein Gel imaging with the UVP GelStudio. All gels were imaged using a 302 nm UV transilluminator. SYBR Green, Gel Green, and SYBR Gold were imaged using AJ emission filter 38-0352-01. Ethidium bromide and SYPRO Ruby were imaged using AJ Filter 38-0220-01 and SYPRO Ruby were imaged using AJ Filter 38-0341-01. All images were pseudocolored to reflect the emission wavelength. All image processing was completed on Analytik Jena’s VisionWorks Data Acquisition and Analysis software version 9.0.

References and Further Reading

1. Ethidium Bromide - US. Available at: https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/nucleic-acid-gel-electrophoresis/dna-stains/etbr.html. (Accessed: 1st August 2019)
2. Chowdhury, A. R. et al. The Killing of African Trypanosomes by Ethidium Bromide. PLOS Pathogens 6, e1001226 (2010).
3. Sharp, P. A., Sugden, B. & Sambrook, J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis. Biochemistry 12, 3055–3063 (1973).
4. Beer, L. A. & Speicher, D. W. Protein Detection in Gels Using Fixation. Curr Protoc Protein Sci CHAPTER 10, Unit-10.5 (2002).
5. Winkler, C., Denker, K., Wortelkamp, S. & Sickmann, A. Silver- and Coomassie-staining protocols: Detection limits and compatibility with ESI MS. Electrophoresis 28, 2095–2099 (2007).

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