GLYCOPROTEIN STAINING
Glycoproteins were stained using PAS Schiff's reagent.
Materials
1. 1% periodic acid in 7.5% acetic acid in distilled water.
2. Schiff's reagent
a) 1.0 gm of basic fuchsin was dissolved in 200 ml of boiling distilled water,
stirred for 5 min and cooled to 5°C and filtered.
b) To the filtrate 20.0 ml of 1N HCl was added, cooled to 20°C before adding
1.0 gm of sodium or potassium metabisulphate. This solution was kept in
dark for 12-24 hr.
c) 2.0 gm of activated charcoal was added and mixed for 1 min, filtered and
stored at room temperature. 5.0 ml of 2 N HCl was added. An aliquot should
not turn red when dried on slide, the reagent discarded when turned pink.
3. 0.5% of sodium metabisulphate in 0.1 N HCl.
Procedure
EGFR was run on 7.5% SDS-PAGE. After the run, the gel was washed continuously
with 2.6 L of 40% methanol and 7% acetic acid overnight. The solution was
changed and the elution continued for 8 hr or the gel was put in 7.5% acetic acid
and kept in RT for 1 hr. The gel was transferred to a tank containing 1% periodic
acid, kept immersed for 1 hr in dark at 4°C. The gel was washed in 7.5% acetic acid
for 10 min and the washing repeated 6 times. After washing the gel was incubated in
Schiff's reagent at 4°C in dark for 1 hr, washed in 0.5% sodium metabisulphate.
The gel was preserved in 7.5% acetic acid.
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40% methanol and 7% acetic acid – 1hr.
7.5% acetic acid - 1 hr (RT)
1% periodic acid – 1hr (4oC, dark)
7.5% acetic acid for 10 min and the washing repeated 6 times
Schiff's reagent - 1hr (4oC, dark)
Wash in 0.5% sodium metabisulphate