Detect Released Oligosaccharides Using The Eagle Eye^® II Still Video Imaging System

Fast imaging and software flexibility

Detect Released Oligosaccharides Using the Eagle Eye^® II Still Video Imaging System

Angela R. Downs • Deborah A. Greer • Stan Ivey
Department of Biology, Delaware State University, Dover, DE

We compared the Eagle Eye ^® II still video system and the Glyko^® FACE ^® SE2000 gel imager system for imaging Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) gels. The data generated from either system were not significantly different, indicating that successful FACE gel analysis does not require a dedicated single-use device (such as the Glyko SE2000). Consequently, the versatile Eagle Eye II still video system is superior because it can be used for many applications without compromising experimental results.

Glycoprotein carbohydrates play important roles in the biological processes of glycosylated proteins.¹ However, analyzing glycan moieties can be an arduous process, given the small recovery amounts, impurity, and low molecular mass of released glycans. In a recent advance, gel electrophoresis has been used to separate fluorophore-labeled oligosaccharides (FACE) released from glycoproteins using endoglycosidases.²

Following electrophoresis, CCD cameras are widely used for all types of fluorescent or colorimetric gel documentation. The Eagle Eye II still video system is designed to visualize, document, and provide analytical tools for any type of DNA or protein gel. In contrast, the Glyko SE2000 gel imager is designed to image only FACE gels.

We compared a FACE gel imaged first using Stratagene’s Eagle Eye II still video imaging system (with a SYBR^® Green band-pass filter), then imaged the gel again using Glyko’s SE2000 gel imager (Figure 1). We then evaluated the time required to image the gel, the ease of use, and the image clarity for both systems.

Sample Preparation

Glycoproteins were chosen to give a reasonable sample of oligosaccharide profiles—including the number of detectable bands, band intensity, and band size—using a Bio-Rad^® N-linked profiling gel for separation. We acquired the glucose ladder, E4 (maltotetraose), and E5 (trypsin inhibitor) standards from Bio-Rad’s N-linked oligosaccharide profiling kit. Transferrin, fetuin, and carboxypeptidase Y were taken from Boehringer Mannheim’s DIG Glycan Differentiation kit. The rabbit polyclonal antibody (IgG) was raised against human P-glycoprotein in our lab. Sample proteins were treated with PNGase F (Bio-Rad kit) to release all N-linked oligosaccharides, following the kit’s instructions. All glycans were labeled with ANTS using reductive amination (Bio-Rad kit procedure). The FACE/ANTS gel fluorophore-tagging method is previously described.³

Oligosaccharide PAGE

 The gel used came preformed in the Bio-Rad N-linked profiling kit. The gel contained 8-aminonaphthalene-1,3,6-trisulphonate (ANTS⁴)-labeled, N-linked oligosaccharides, released from various glycoproteins, separated by oligosaccharide PAGE. The gel was run in a Bio-Rad mini PROTEAN II gel apparatus with the kit’s oligosaccharide electrophoresis running buffer. The gel was run at 4ºC under a constant current of 15 mA for one hour. Immediately thereafter, the gel plate was cleaned, wiped dry, and imaged using the Eagle Eye II still video system. Then the gel was cleaned again and imaged using Glyko’s gel imager. Both gel images were taken within 10 minutes of the elapsed time after the gel run.

Imaging the Gels

The gel was first imaged using the Eagle Eye II still video system. To position and focus the gel, only the white light from Stratagene’s dual-light transilluminator was used. The bands in the profiling gel (Figure 1, Panel A) were excited using the overhead 312-nm light that comes with the Eagle Eye II unit. The UV lights from the transilluminator were not used. The lens aperture was set at an f-stop of 1.2 and was equipped with Stratagene’s SYBR Green band-pass filter. A negative image was created using dynamic integration at 25 frames/0.8 seconds, with incremental counts of 25 frames/0.8 seconds for 9.6 seconds.

Next, the gel was imaged using Glyko’s SE2000 gel imager (Figure 1, Panel B), which is similar to Bio-Rad’s Glyco Doc Imager. An image rectangle was used to match the outline of the gel that was imaged using the Eagle Eye II still video system. Saturation was set at 0.0%.

Conclusions

Using Stratagene’s Eagle Eye II still video system, it takes less than 5 minutes to image a FACE gel and produces comparable results to those obtained with the Glyko SE2000 gel imager.

We discovered that the SYBR Green filter and the overhead 312-nm UV bulb must be attached for oligosaccharide profiling gels because the dual-light UV transilluminator (312 nm) was too intense for the gel’s fluorophore-labeled oligosaccharides: it becomes quickly bleached from the multiple bulbs in the UV lightbox. We also found that the gel must be imaged within 15 minutes of the gel run for optimum band sharpness; after the same amount of time at room temperature, the bands spread significantly.

While the contrast in banding patterns appears higher in the gel image taken using Glyko’s gel imager, this is probably due to the Glyko camera’s shorter focal distance. However, the same banding numbers and definitions are seen in both gels. Additionally, Stratagene’s gel analytical software program can be customized to analyze and quantify the separated oligosaccharide bands in the oligosaccharide profiling gel.

REFERENCES

1. Dwek, R.A. (1995) Biochemical Society Transactions 23: 1-25.

2. Fukuda, M. and Kobata, A. (1993) In Glycobiology: A Practical Approach. IRL Press by Oxford University Press, Inc., New York.

3. Jackson, P. (1994) Analytical Biochemistry 216: 243-252.

4. Jackson, P. (1993) Biochemical Society Transactions 21: 121-125.