( to image the gel the ease o...)Detect Released Oligosaccharides Using the Eagle Eye II,,,Still Video Imaging System

Sample Preparation

Glycoproteins were chosen to give a reasonable sample of oligosaccharide profilesincluding the number of detectable bands, band intensity, and band sizeusing a Bio-Rad N-linked profiling gel for separation. We acquired the glucose ladder, E4 (maltotetraose), and E5 (trypsin inhibitor) standards from Bio-Rads N-linked oligosaccharide profiling kit. Transferrin, fetuin, and carboxypeptidase Y were taken from Boehringer Mannheims 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 kits 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 kits oligosaccharide electrophoresis running buffer. The gel was run at 4C 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 Glykos 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 Stratagenes
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