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 100 x lower concentrations
than Coomassic Brilliant Blue (i.e., femtomole levels of protein).
The sensitivity of silver staining is 2 to 5 ng/protein band.
MATERIALS AND SOLUTIONS
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
Developer (525 ml)
Developing Solution --------------------------- 25 ml
Deionized, distilled H2O ----------------------- 500 ml
PROCEDURES
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.
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.
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.
NOTES
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.
REFERENCES
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.