EDC Impressum & DSE

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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Special Staining for ‘Oligo IgGs’: Ammoniacal Silver Stain

Juli 13, 2018

Ammoniacal Silver staining:
Special IEF-staining for basic proteins (f.e. IgG)
Attention: 1. Fixing solutions containing Acetic Acid and MeOH (or EtOH) have to be prepared freshly!
                2. Do not use plastic-trays, plastic-beakers etc., use only glas and/or stainless steel

step

reagent 

volume

gel>0.5 mm

gel<0.5 mm

1 Fixing

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

200 ml

45 min

20 min

2. Washing

H2O dist

200 ml

5 min

5 min

3. Rinsing I

50 % methanol / 10% acetic acid (v/v) (ethanol also works!) *
(add 100 ml MeOH and 20 ml HAc and make up to 200 ml with distilled water)

200 ml

40 min

20 min

4. Rinsing II

5% methanol / 7% acetic acid (ethanol also works!) *
(add 10 ml MeOH and 14 ml HAc and make up to 200 ml with distilled water)

200 ml

20 min

20 min

5. Incubation

2.5 % glutardialdehyde *
(add 20 ml Glutardialdehyde (25%) to 180 ml with distilled water)

200 ml

20 min

15 min

6 - 9. Washing

H2O dist
(optional: Hold overnight in the last step = best results)

4 (3) x 200 ml

4 x 20 min

3 x 20 min

10. Silvering
(Prepare freshly before use!)

0.125% AgNO3, 0.02 M NaOH, 0.2% Ammoniak *
 (to 50 ml distilled water add 4 ml 1M NaOH, then add 1.7 - 2.0 ml ammonia-solution (25%) and slowly add 5 ml of the silver-solution (5%) *

200 ml

40 min

30 min

11-12. Washing

H2O dist

2 x 200 ml

1 + 5 min

1 + 5 min

13. Developing
(Prepare freshly before use!) 

0.0025% citric acid + 0.02% formaldehyde *
(take 1 ml of the citric acid-solution (0.5%) and make up to 200 ml with distilled water, add 100 ul of the formaldehyde (37%)

200 ml

3-5 min
(visual control)

3-5 min
(visual control)

14-16. Stopping & Preserving

10% Ethanol, 1% Acetic Acid, 5% Glycerol *
 (add 60 ml EtOH + 30 ml Glycerol + 6 ml HAc and make up to 600 ml with Bidest)

3 x 200 ml

3 x 10 min

3 x 10 min

Stock Solutions
1. 20% TCA: (Merck 1.00807.1000) Fill up this bottle with Bidest (till the upper curvature of the Merck-bottle, about ~700 ml), solution process takes overnight. This solution is stable for months!
This gives a “100%” solution; 100 ml + 400 ml Bidest = ~20%
2. 5% AgNO3: (Merck 1.01512.0100) Weigh in 5 g in 100 ml Bidest, fill in a brown bottle! Stable solution.
3. 1M NaOH: (Merck 6462.1000) Weigh in 4 g in 100 ml Bidest.
4. 0.5% Citric Acid: (Merck 1.00244.1000) Weigh in 50 mg in 10 ml Bidest, only 1 week stable! *


Chemicals
Methanol, Ethanol, Acetic Acid, Ammonia (25%), Formaldehyd (37%) and Glycerol are from Merck.

Remarks
* Prepare freshly!

* Any brownish precipitate should immediately disappear, if not: the ammonia-solution is too old!

Droplet-Test

The droplet-test should show the “colour-kaleidoscope”: within the first 5 seconds three colours should show up: green - brown - yellow