Silver Staining of Sulfated Glycosaminoglycans: A Staining Method for Visualization of Sulfated Glycosaminoglycans in Polyacrylamide Gels

1. Silver staining protocol

1. Prepare all solutions necessary for silver staining in advance (Table 1).
   NOTE: Do not directly touch the PAGE gel until it has been stained, developed, and placed in stop solution. Instead, manipulate the gel using clean plastic or glass tools. Directly handling the gel will result in finger-print distortions and other visible artifacts on the gel after staining.
2. Once the run is completed, disassemble cassette and extract gel in a clean, medium-large container filled with deionized, filtered water.
   NOTE: To avoid directly handling the gel, use pipette tip or other plastic object to gently peel the gel away from the cassette while submerged in water. Gel may be fragile – handle carefully.
   1. Discard the water. Stain the gel in Alcian blue staining solution for 5 min.
   2. Discard Alcian blue stain. Quickly rinse/wash 2-3x with deionized, filtered water until most of the Alcian blue staining solution has been removed.
   3. Allow to de-stain in deionized, filtered water overnight on rocker. Ensure that there is ample volume of deionized, filtered water to ensure any residual stain is fully washed off the gel overnight.
   4. Wash gel in 50% methanol (40 min total, change solution 2-3x).
   5. Wash gel in deionized, filtered water for 30 min. Discard water and repeat 3 more time for a total of 2 h, replacing the water each time.
   6. In a fresh, clean container, stain the gel for 30 min in silver nitrate staining solution.
   7. Quickly rinse/wash 2-3x in deionized, filtered water to fully remove the silver staining solution.
   8. Wash for 30 min in deionized, filtered water. Discard water and repeat 2x for a total of 90 min, replacing the water bath each time.
   9. Discard water and add developing solution.
   10. Once developing solution is added, carefully observe the gel and watch for the appearance of bands. Depending on the quality of the stain and the mass of the sample loaded, development can take anywhere from a few seconds to several minutes.
   11. As soon as the desired bands are visible, immediately discard developing solution and wash briefly with stop solution.
   12. Discard the stop solution wash and replace with fresh stop solution. Allow to soak for 1 h on a rocker or shaker.
   13. Wash in deionized, filtered water overnight (however, the gel can be imaged immediately after stop solution wash).

Table 1: Solutions required for silver staining of glycosaminoglycans separated by polyacrylamide gel electrophoresis. All solutions must be filtered (0.22 μm) before use.