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

Protocol

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.

NOTE: All solutions must be filtered (0.22μm) before use.
Solution	Recipe	Composition	Comments
Alcian blue staining solution (500mL)	Alcian blue 8GX powder	2.5g	Desired concentration: 0.5% w/v
	2% v/v glacial acetic acid	500mL
Silver stain (200 mL)	Deionized water	192.7 mL	Prepare stain solution fresh; use within one week.
	7.6M sodium hydroxide	2 mL	To make, add 3.04g sodium hydroxide pellets to 10 mL water
	Ammonium hydroxide	3.3 mL
	4M silver nitrate solution	2 mL	To make, add 3.397 g silver nitrate to 5 mL water. Add dropwise while stirring to avoid precipitation.
Developing solution (501 mL)	Deionized water	500 mL	Developing solution must be made fresh and used within 24 h.
	2.5% w/v citric acid	1 mL	To make, add 100mg to 4 mL deionized water. Citric acid must be made fresh.
	Formaldehyde	250μL
Stop solution (500 mL)	Deionized water	300 mL
	Glacial acetic acid	20 mL
	Methanol	90 mL

Materials

Name	Company	Catalog Number	Comments
Alcian Blue 8GX (solid)	ThermoFisher Scientific	AC400460100
Ammonium hydroxide (liquid)	ThermoFisher Scientific	A669S-500	Certified ACS
Barnstead GenPure Pro Water Purification System	ThermoFisher Scientific	10-451-217PKG	Any water deionizing/ purification system is an acceptable substitute
Glacial acetic acid (liquid)	ThermoFisher Scientific	A35-500	Certified ACS
Methanol (liquid)	ThermoFisher Scientific	A412-500	Certified ACS
Silver nitrate (solid)	ThermoFisher Scientific	S181-25	Certified ACS
Sodium hydroxide (solid)	ThermoFisher Scientific	S392-212
Vacuum filter unit, single use, 0.22uM pore PES, 500mL volume	ThermoFisher Scientific	569-0020	Alternative volumes and filter materials acceptable
Molecular Imager Gel Doc XR System	Bio-Rad	170-8170	Any comparable gel imaging system is an acceptable substitute