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.
|  |