Molecular Techniques and Methods

Silver Staining of Proteins in PAGE

Copy Right 2001/ Institute of Molecular Development LLC


Silver staining relies on differential reduction of silver ions which is the basis for photographic processes. A highly sensitive photochemical silver staining technique permits the detection of polypeptides in gels at more than 100x lower concentrations than Coomassic Brilliant Blue (i.e., femtomole levels of protein).

The sensitivity of silver staining is 2 to 5 ng/protein band.


Fixing Solution (100 ml)
50% Methanol ------------------------------- 50 ml of 100% Methanol
10% Acetic acid ----------------------------- 10 ml of Acetic acid
Deionized, distilled H2O ---------------------- 40 ml

Use Fixing Solution only once.

Destaining Solution (100 ml)
5% Methanol --------------------------------- 5 ml of 100% Methanol
7% Acetic acid ------------------------------- 7 ml of Acetic acid
Deionized, distilled H2O ---------------------- 88 ml

Silver Nitrate Solution (208 ml)
0.5% NH4OH -------------------------------- 3.5 ml of Conc. NH4OH (30%)
0.08% NaOH -------------------------------- 0.35 g of NaOH pellet
Deionized, distilled H2O ---------------------- 196.5 ml

Mix with a magnetic stirrer.

Add slowly 8 ml of 19.4% (1.6 g/ 8 ml) Silver nitrate.

If the solution is cloudy, carefully add Conc. NH4OH until it clears.

The solution should be used within 20 min.

Developing Solution (100 ml)
17 mM Sodium citrate -------------------------- 1.7 ml of 1 M Sodium citrate
37% Formaldehyde Solution -------------------- 0.5 ml
Deionized, distilled H2O ------------------------- 99 ml

l        For LC-MS/MS analysis, do not add formaldehyde.

l        Formaldehyde cross-links proteins to the gel matrix, resulting in low extraction efficiency.

l        Replace Formaldehyde by Carbohydrazide for LC-MS/MS analysis.

l        Pierce SilverSNAP Stain Kit II (24612) contains formaldehyde in Enhancer. Do not use Enhancer for LC-MS/MS.


Developer (525 ml)
Developing Solution --------------------------- 25 ml
Deionized, distilled H2O ----------------------- 500 ml


1. Place the polyacrylamide gel in a plastic box on an orbital shaker and fix in the Fixing Solution for 30 min.

2. Fix the gel in Destaining Solution for 60 min.

3. Fix the gel in 10% Glularaldehyde for 30 min.
* Skip this step for LC-MS/MS. Aldehyde cross
-links proteins to the gel matrix, resulting in low extraction efficiency.

4. Wash gel with deionized, distilled H2O for 30 min. Repeat 4 times.

5. Stain the gel with Silver Nitrate Solution for 15 min with vigorous shaking.

6. Transfer the gel to another plastic box and wash with deionized, distilled H2O for 1 min.

Repeat 5 times.

7. Transfer the gel to another plastic box.

Add Developer, and shake vigorously until the bands appear as intense as desired.

If the Developer turns brown change to fresh Developer.

8. Transfer the gel to Kodak Rapid Fix for 5 min.

9. Wash gel exhaustively in water to remove Rapid Fix.

10. To maintain a permanent gel record, the gel may be dried.

Place the gel between 2 cellophane membrane backing sheets which are slightly larger than the gel. Place this sandwich between 2 sheets of Whatman 3MM filter paper and dry in a gel dryer at 80oC for 60 min.


The high sensitivity of the silver staining technique renders it susceptible to impurities and staining artifacts. It is mandatory that the polyacrylamide gels and all staining solutions be prepared from high quality reagents in order to avoid staining artifacts. Especially important is the use of high quality water (distilled and deionized, carbon-filtered H2O).

The glassware used for gel polymerization and the plastic staining boxes should be cleaned thoroughly, and gels should be handled with vinyl, powder-free gloves. To avoid uneven staining of the gel surface, the polyacrylamide gel should be covered with a sheet of Parifilm in order to uniformly wet the gel surface during staining, and touched only very gently with gloved hands.


Merril CR, Goldman D, Van Keuren ML (1984) Gel protein stains: Silver stain. Meth. Enzymol. 104: 441-447.

Oakley BR, Kirsch DR, Morris NR (1980) A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105: 361-363.

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