Protein Protocols Protein Protocols

Glycoprotein Detection 765
3.1.1. Gel Staining (see Note 1)
1. Soak the gel in solution C for 30 min (see Note 2).
2. Wash in distilled water for 10 min. All of the ethanol must be removed from the gel, so
make sure that the gel is immersed in the water properly. If necessary wash a second time
to ensure the removal of the ethanol.
3. Incubate in solution A for 30 min. Beware of the fumes from the acid. From this point
onwards the gel should be placed in the fume hood.
4. Wash in distilled water for at least 6 × 5 min or 5 × 5 min and 1× overnight.
5. Wash in solution B for 2 × 10 min. At this stage make up 100 mL of solution B and
perform 2 × 30 mL washes; save the final 40 mL for step 7.
6. Incubate in Schiff’s reagent for 1 h in the dark. It is essential after adding the Schiff’s
reagent that the gel is kept in the dark, as any light will stop the color from developing.
7. Incubate in solution B for 1 h in the dark.
8. Wash several times in solution D for a total of at least 2 h and leave as long as overnight to
ensure good color detection (see Notes 3 and 4).
9. Store the gel in solution E.
3.1.2. Membrane Staining (see Note 5)
1. Wash the membrane in distilled water for 5 min (see Note 6).
2. Incubate in solution A for 30 min.
3. Wash in distilled water for 2 × 5 min.
4. Wash in solution B for 2 × 5 min.
5. Incubate for 15 min in Schiff’s reagent (see Note 3).
6. Wash in solution B for 2 × 5 min.
7. Air dry the membrane.
3.2. DIG–Anti-DIG AP Labeling (See Note7)
This staining procedure can only be used on membranes; however, the proteins can
be prelabeled in solution before electrophoresis, or labeled on the membrane after blotting
(see Notes 8 and 9). In both cases the color development is the same. Nitrocellulose
membranes can be used but some background staining can occur with postlabeling.
In preference, the proteins should be blotted onto PVDF.
The reagents for this method are provided in the Roche Diagnostics DIG Glycan
Detection Kit and the methods described are essentially taken from that kit.
A similar kit based on biotin/streptavidin binding instead of the DIG–Anti-DIG
interaction is marketed by Bio-Rad as the Immun-Blot ® Glycoprotein Detection Kit.
3.2.1. Prelabeling (see Note 8)
1. Dilute protein solution 1:1 to 20 µL with buffer B (see Note 7).
2. Add 10 µL of solution 1 and incubate for 20 min in the dark at room temperature.
3. Add 10 µL of solution 2 and leave for 5 min. The addition of the sodium bisulfite destroys
the excess periodate.
4. Add 5 µL of DIG-succinyl-e-amidocaproic acid hydrazide, mix, and incubate at room
temperature for 1 h. Sensitivity may be increased by increasing the incubation time to
several hours.
5. Add 15 µL buffer D and heat the mixture to 100°C for 5 min to stop the labeling.
6. Separate the labeled glycoproteins by SDS-PAGE and blot to the membrane (see Note 10)
which is now ready for the staining reaction (see Subheading 3.2.3.).
3.2.2. Postlabeling (see Note 9)
1. Wash the membrane for 10 min in 50 mL buffer E (PBS). (see Note 11).