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Protocol for PAS staining of Glycoproteins
（Modified.Apr.2004）
Materials
1. Reagent A:400ml methanol,70ml acetic acid,530ml destilled water.
2. Reagent B:75ml acetic acid,925ml destilled water.
3. 1% periodic acid in 7.5% acetic acid in distilled water.
4. 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.
5. 0.5% of sodium metabisulphate in 0.1 N HCl.
Procedure
1. Perform SDS-PAGE in slab gels.After the run, the gel was washed continuously with Reagent A overnight. (This step leaches out the SDS)
2. 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.
3. The gel was transferred to a tank containing 1% periodic acid, kept immersed for 1 hr in dark at 4°C.
4. The gel was washed in 7.5% acetic acid for 10 min and the washing repeated 6 times. (This amber color will fade as iodide is formed from iodine in approximately 1hr)
5. After washing the gel was incubated in Schiff's reagent at 4°C in dark for 1 hr, washed in 0.5% sodium metabisulphate. (Staining with Schiff’s reagent is performed by submersing the gel in this solution and allowing the pink color to develop in the dark on ice.It is possible to leave the sample at this point in a dark cold room overnight to examine in the morning.Color development is gradual,and stained bands are stable for serveral days)
6. The gel was preserved in 7.5% acetic acid.
Reference:
1. Craig Gerard.purification of glycoprotein.Methods in enzyme.1990,Vol.182.529-39.
