silver staining, silver stain

Silver development protocol (silver stain) for 0.75mm polyacrylamide gels
If you're thinking of using thicker gels - don't

Caution: Avoid thiols; they release peptides from keratin creating "rain". For protein gels reduce and alkylate samples (with 5mM DTT [see advice on DTT], and 15mM iodoacetamide respectively)

1- Agitate gel twice with 50% MeOH, 10% acetic acid (Fixer 1) for 15min ea. Gels can be held for several days in Fixer 1.

2- Agitate in 10% EtOH, 5% glacial acetic acid (Fixer 2) for 6min. We use commercial (cheap) alcohols.

3- Agitate in distilled water. Agitate twice (9 min each) with 500ml DW. Thorough rinsing gives a low, uniform background.

4- Agitate in 500ml of 20mg/L Na2S2O4 (hydrosulfite [dithionite], make fresh) in distilled, deionized water (DDW) for 9min (sensitization). Caution - the half-life of dithionite solution is tens of minutes.

5- Pour off solution; w/o rinsing add 200ml, 0.1% AgNO3 (200mg) in DDW add 150ul 37% formaldehyde. Agitate for 9min.

6- Rinse 30 sec with DDW to remove excess AgNO3; add 200ml image developer (1ml 37% formaldehyde per liter of 3% sodium carbonate) mixed with 100ul 10g/L sodium thiosulfate, Agitate to desired staining intensity (3 to 6min). Thiosulfate complexes silver preventing silver carbonate precipitation.

7- Pour off developer, add 80 ml of stop solution (50g tris, 25ml glacial acetic per liter of distilled water).

8- Store gel in 10% glycerol with bacteriostat (0.02% NaN3).

References: Morrissey, J.H. (1981). Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Analytical Biochem 17, 307-31. Rabilloud, Thierry (2000) Detecting proteins separated by 2-D gel electrophoresis. Analytical Chemistry 72, 48a-55a.


The protocol is robust. Occasional slow development appears to correlate with old formaldehyde. Doubling the formaldehyde concentration in the developer solves the problem.

What to look for in a silver stain protocol

Avoid silver-ammine stains. Ammonia is touchy stuff and procedures are lengthy. Glutaraldehyde is unnecessary, unless you've got basic proteins (luteovirus capsids? - no data). Be sure gel is thin (0.75mm). Stain should incorporate a sensitizer (dithionite in my case) and a silver chelator (thiosulfate). The latter prevents silver carbonate precipitation. Use high quality distilled water in the last stages of development. Otherwise technical grade chemicals (alcohols, glycine, etc) work fine. I've not successfully stained tricine gels - even with extensive washing; I presume tricine is a better silver precipitant than glycine.