Silver stain

MS/MS compatible protocol. Click here for references using this protocol.

Suitable for 1D and 2D PAGE. Volumes should be scaled according to the experiment.


1.Fixation:

a. 30' in 5% acetic acid + 50% methanol.  x2
b. 30' in  in 50% methanol.
c. Let the gel to rehydrate in water for at least 2 hours with changes every 30'.


2. Sensitation.

a. 1' in 100 mM (= 20mg/100ml) Sodium Thiosulphate 5-hydrate  (Na2S2O3•5H2O).
b. Brief washes with water.
c. Wash for 1' with water.  x2
d. Brief washes with water.


3. Silver.

a. Incubate at 4 degrees for 30' with pre-cooled 6 mM (=100mg/100ml) silver nitrate (AgNO3).
b. Brief washes with water.
c. Wash for 1' with water.  x2
d. Brief washes with water.


4. Developing.

190 mM (2g/100l) sodium carbonate (Na2CO3) + 0.0074% formalin (CH2O) until the bands started to appear.


5. Stop.

5% acetic acid.


6.Storage.

After 10' in stop buffer, store in water at 4 degrees.







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