Protein Protocols Protein Protocols

Glycoprotein Detection 769
5. Resuspend in 25 µL and add 0.5 nmol lactobionic acid as internal standard for quantitation.
Analyze by HPAEC-PAD using a linear gradient of 0–200 mM NaAcetate in 250 mM
NaOH for 30 min (see Notes 24–26).
6. Compare retention time with standard of 1 nmol N-acetyl and N-glycolylneuraminic acids.
3.5.2. Analysis of Neutral Monosaccharides
1. Add 100 µL of 2 M TFA to the desialylated membrane spots (see Note 27). Mix and close
tube (see Note 24). Re-wet membrane with methanol before addition of acid if dried out.
2. Incubate in a boiling water bath for 4 h.
3. Remove membrane from tube and wash with 50 µL of water. Keep membrane for subsequent
amino acid analysis if required.
4. Dry combined solutions in a Speed Vac concentrator.
5. Resuspend in 25 µl of water and add 0.25 nmol of 2-deoxyglucose as internal standard for
quantitation. Analyze by HPAEC-PAD eluted isocratically with 12 mM NaOH for 30 min
(see Notes 24–26).
6. Compare with a standard mixture containing fucose, 2-deoxyglucose, galactosamine, glucosamine,
galactose, glucose, and mannose (5 µl of 1 mM solution) (see Note 28). This is
the order of elution of the monosaccharides from the CarboPac column.
4. Notes
4.1. Periodic Acid/Schiff Staining
1. This method can be used on PAGE gels, agarose, or polyacrylamide–agarose composite
gels. The procedure should be carried out on an orbital shaker or rocker (an orbital shaker
may cause background swirls on some gels). High background staining can occur when
staining some batches of agarose or composite gels.
2. All steps in this procedure should be performed with shaking and should be carried out in
a fume hood.
3. A negative result often means that insufficient protein is present or that the protein has
little glycosylation
4. Be aware that highly glycosylated proteins do not transfer well to nitrocellulose membranes,
and failure to detect may be due to inefficient transfer to the membrane. It is
sometimes useful to carry out the stain on the gel after transfer, to test for remaining
glycoprotein.
5. PAS staining after transfer of glycoproteins to membranes eliminates the need for extensive
fixation steps, as well as shortening the time needed for washing steps , without loss
of staining intensity. Results are easier to visualize and are easier to store. Before Western
transfer, it is suggested to gently rock the gel for 30–60 min in transfer buffer so as to
remove SDS, which can lead to higher backgrounds during membrane staining.
6. Periodic acid–Schiff staining on nitrocellulose membranes increases sensitivity slightly
and reduces washing times considerably compared with gel staining.
4.2. DIG–Anti-DIG AP Labeling
7. All steps in the procedure except for color development should be done on a shaker or
rocker. Swirling background staining may occur when using a orbital shaker.
8. Prelabeling gives a higher sensitivity than postlabeling, requires less DIG (1 µl per labeling),
and can be used with