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Electrophoresis
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Dr. Hanspeter Schickle
Wolfgang Gstrein

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IEF-Silver Staining Protocols: Alkaline Silver Stain

Juli 13, 2018

Alcaline Silver staining:

General IEF Staining for acidic and neutral proteins.

Special silver stain for basic proteins (eg. IgG): “Ammoniacal Silver Stain

step

reagent 

volume

gel>0.5 mm

1 Fixing

20% trichloroacetic acid (w/v)
(40 ml “100%” TCA-solution in 200 ml)

200 ml

45 min

2 Washing

H2O dist.

200 ml

5 min

3 Rinsing #1

50% ethanol/ 10% acetic acid (v/v)
(100 ml EtOH / 20 ml HAc in 200 ml)

200 ml

40 min

4 Rinsing #2

5% ethanol/ 7% acetic acid (v/v)
(10 ml EtOH / 14 ml HAc in 200 ml)

200 ml

20 min

5 Incubation

165 mM Sodium Acetate; 2.5 % Glutardialdehyde (w/v)
0.07% (w/v) Benzenesulfonic acid
(4.5 g NaAc / 20 ml Benzenesulfonic stock sol.,
20 ml Glutardialdehyde (25%) in 200ml)
 

200 ml

20 min

6 Washing

H2O dist.

3 x 200 ml

3 x 20 min

8 Silvering

0.2% AgNO3 /0.05% Formaldehyde (w/v), 0.07% Benzenesulfonic Acid
(8 ml AgNO3 sol., 20 ml Benzenesulfonic acid sol., 260ul HCHO (37% w/v) per 200 ml)

200 ml

40 min

9 Washing

H2O dist.

3 x 200 ml

3 x 1 min

10 Developing

2.5 % Na2CO3 / 0.05% Formaldehyde
(5g Na2CO3, 260ul HCHO (37% w/v) per 200 ml)

200 ml

2 -3 min

11 Stopping
Preserving

10% HAc, 5% Glycerol
(20 ml HAc + 10 ml Glycerol)

200 ml

30 min

Drying

air dry , on the support-film

---

overnight

droptest

Droplet-test: within 3 seconds

Stock Solutions
1. 20% TCA: (Merck 1.00807.1000) Fill up this bottle with (about 850 ml) Bidest. This gives a “100%” solution.
2.  Benzenesulfonic (Merck 8.00468.0250), 0.7 % (10-fold solution) 1,4 g / 200 ml
    ---> 100 + 400 Bidest = 20%.
3. AgNO3: 5 % (20-fold) solution, 5 g in 100 ml.
4. Na2CO3: 25% (ten-fold) solution, 25 g (water-free!) in 100 ml.

AgNO3, Na2CO3 (water-free), Sodiumthiosulfate are from Merck
Ethanol, Acetic Acid, Formaldehyd (37%), Glutardialdehyde, Citric Acid and Glycerol are from Merck

Abbr.: HAc = Acetic Acid, NaAc = Sodium Acetate