Protein Protocols Protein Protocols

858 Brandley, Klock, and Starr
Fig. 1. Profiles of N-linked oligosaccharides from several glycoproteins. ANTS-labeled oligosaccharides
released by peptide N-glycosidase (PNGase F) from six different glycoproteins
are shown. Lane 1 contains an oligosaccharide ladder standard of partially hydrolyzed wheat
starch with G4 representing glucotetraose; lane 2, chicken trypsin inhibitor; lane 3, bovine
fetuin; lane 4, human α-acid glycoprotein; lane 5, bovine ribonuclease B; lane 6, human chorionic
gonadotropin (hCG); and lane 7, chicken ovalbumin. The profiles show a wide variety of
different glycosylation patterns, indicating the minimum number (some oligosaccharides will
comigrate) of different types of oligosaccharide present and their relative quantities. The images
presented in this review were obtained by imaging the gels following electrophoresis using a
CCD (charge-coupled-device)-based imaging system (Glyko).
3.8. Gel Handling
1. If the gel is no longer needed it should be properly discarded.
2. Following imaging of the oligosaccharide gels, the glass plates can be separated and the
gels dried on a flat bed gel drier between sheets of Teflon membrane at 80°C for 1 h.
After the gel is dry, carefully peel the Teflon sheets away from the gel. Gels dried in this
manner can be stored indefinitely in a dark dry location and can reimaged at any time with
minimal bleaching.
3. Following imaging, the gel cassette can be placed back in the electrophoresis apparatus
and the run continued in order to improve the resolution of the oligosaccharide bands. In
this case, the upper buffer should be saved and reused until the run is finally terminated.
Note that diffusion of carbohydrate bands and subsequent poor resolution will occur if
the time between electrophoresis and imaging exceeds 15 min.
4. Notes
1. The glycoprotein sample should ideally first be dialyzed against distilled water and stored
lyophilized in a 1.5 mL microfuge tube. If the sample needs to be in a buffered solution,
one can place the sample in 50 mM sodium phosphate buffer, pH 7.5, at a final concentration
of at least 100 µg/50 µL or 2 mg/mL. Best results are obtained if the total salt concentration
of the solution is