Silver Staining Kit
catalogue Number 30-41-10
Proteins and Peptides can be visualised after separation by SDS Poly-acrylamide Gel electrophoresis,
using silver salts. The method described uses Silver Nitrate which is very sensitive and can detect 0.1-1.0ng of
Protein/ Peptide. The process relies on the reduction of silver ions by amino acids which bind to side chains of
proteins and peptides. (Switzer et al 1979, Oackley 1990, Sammons et 1981) this method is described in
Maniatis, Fritsch and Sambrook Molecular Cloning Handbook volume III 2nd Edition.
Separate Proteins and Peptides by SDS Poly-acrylamide Gel Electrophoresis and using Fixative Solution 1
(Catalogue Number 30-41-SS1) to fix the Proteins and Peptides within the gel. Fix for 4-12 hours at room
temperature with gentle intermittent shaking. The solution contains Ethanol Glacial Acetic Acid, water.
Discard the fixing solution and add solution 2 (Catalogue Number 30-41-SS2) use a sufficient quantity and
incubate the gel at room temperature for 30 minutes. Repeat step 2
Discard the ethanol solution 2 and add a copious volume of deionised purified water and wash the gel
thoroughly. The gel will swell slightly during its rehydration. Repeat this step twice.
Discard the water washes and wearing gloves add 0.1% of AgNO3 freshly diluted from Solution 3 (Catalogue
Number 30-41-SS3 brown bottle) which is a 20% stock solution.
Incubate the gel for 30mins at room temperature with gentle shaking.
Discard the 0.1% AgNO3 solution (Diluted silver Nitrate) and wash both sides of the gel using copious quantity
of deionised water or a water stream. Do not allow the gel to dry out otherwise staining artifacts may occur.
Add a quantity of 2.5% Sodium Carbonate Formaldehyde Solution 4 (Catalogue Number 30-41-SS4) and
incubate the gel at room temperature with gentle agitation. Watch the gel carefully as stained bands of
Peptide/Protein will appear within a few minutes, continue until the staining is at a level which is clearly visible
and contrasting. Prolonged incubation leads to a high background silver staining within the gel.
The reaction is therefore quenched by washing the gel with a 1% Acetic Acid Solution 5 (Catalogue Number 3041-SS5) for a few minutes which stops the silver from further oxidation. The gel is then washed with copious
quantities of deionised purified water. Wash the gel for approximately 10 minutes then preserve the gel by
drying.
(Volumes supplied to stain 10cm gels)
NB. Deionised purified water not supplied with kit. (The silver Nitrate supplied is a stock solution and needs to
be diluted to 0.1%v/v with deionised purified water before use. This may be measured out prior to use if not
all the solution is to be used. All other solutions are supplied as working strength.)