2dsilver

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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		2D-Silver Staining Protocols

		This 2D staining recipes are not identical to the normal SDS-silver staining recipe! 1.) Above all, the proteomics-gels should be silver-stained in a way that the mass-spectrometer results will not be disturbed (no Glutardialdehyde!) 2.) These staining recipes should also function with the NF-film supports (no alcoholic solution above 40%, no heat over 50 degrees Celsius!) 3.) EDC’s 2D-gel matrix is different to the one-dimensional matrix (less formaldehyde combined with Sodium-thiosulfate) Film-supported gels should be placed on the tray’s bottom, gel-side up! Large gels should be fixed at the bottom so that they won’t bob up!

	film-supported gels normal size: horizontal 2D-gels 0.65 mm large size: double the volumes, GE Autostainer: use the magnetic bars to hold the large gels down!

		April 28, 2023

step

reagent

volume

steps

temp
(degrees Celsius)

total time

1 Fixing

10% (v/v) Acetic Acid, 40% (v/v) Ethanol, 1% (w/v) Citric Acid
(40 ml Acetic Acid + 160 ml Ethanol + 4 g Citric.Acid in 400 ml)

2 x 200 ml

30 min

ambient

60 min

[SemiCollCoom] [ProtFluoresc]

come in here with the already semi-colloidal Coomassie or fluorescence stained gels:
(destained)

50 (NF-film)
65 (normal-film)

(3 h)

2-5 Washing

H₂O dist

4 x 200 ml

4 x 10 min

ambient

40 min

6 Conditiong*

0.03% (w/v) Sodium-Thiosulphate + 10 mM Sodium Acetate + 30% (v/v) Ethanol
(65 mg Sodium-Thiosulfate + 300 mg NaAc + 60 ml EtOH in 200 ml)

200 ml

30 min

ambient

30 min

7 - 10 Washing

H₂O dist

4 x 200 ml

4 x 5 min

ambient

20 min

11 Silvering

0.25% (w/v) AgNO₃ /0.03% Formaldehyde (v/v)
(500 mg AgNO₃/ 180 ul Formaldehyde (37%) in 200 ml)

200 ml

45 min

ambient

45 min

12-14 Washing

H₂O dist

3 x 200 ml

3 x 1 min

ambient

3 min

15 Developing

2.5 % Na₂CO₃(w/v)_{/ 0.03% (v/v) Formaldehyde / 0.00075% (w/v) Sodium-Thiosulfate}(5 g NaCO₃/ 180 ul Formaldehyde / 75 ul Sodium-Thiosulfate (2%, fresh!) in 200 ml)

200 ml

4 - 6 min
visual control

ambient

5 min

16 Stopping/
Preserving

10% (v/v) Acetic Acid + 10% (v/v) Glycerol
(20 ml HAc + 20 ml Glycerol in 200 ml)

200 ml

20 min

ambient

20 min

17 Drying

air-dry on the support-film overnight
(then roll on a polyester-sheet)

200 ml

16 h

ambient

overnight


	* Gels produced via washed and dried raw products 0.2% (w/v) Sodium-Thiosulphate + 5 mM Benzenesulfonic Acid + 165 mM Sodium Acetate (400 mg Sodium-Thiosulfate + 160 mg Benzenesulfonic Acid + 4.5 g NaAc + 60 ml EtOH in 200 ml)

		film-supported gels large size: vertical 2D-gels 1.0 mm, GE Autostainer: use magnetic bars to hold the large gels down!

step

reagent

volume

steps

temp
(degrees Celsius)

total time

1-2 Fixing

10% Acetic Acid, 40% Ethanol, 1% Citric Acid
(80 ml Acetic Acid + 320 ml Ethanol + 8 g Citric.Acid in 800 ml)

2 x 400 ml

60 min

ambient

120 min

[SemiCollCoom] [ProtFluoresc]

come in here with the already semi-colloidal Coomassie or fluorescence stained gels
(destained and 3 x 15 min H₂O dist)

50 (NF-film)
65 (normal-film)

(4h)

3-6 Washing

H₂O dist

4 x 400 ml

4 x 15 min

ambient

60 min

7 Sensitizing

0.03% (w/v) Sodium-Thiosulphate + 10 mM Sodium Acetate + 30% Ethanol
(130 mg Sodium-Thiosulfate + 600 mg NaAc + 120 ml EtOH in 400 ml)

400 ml

60 min

ambient

60 min

8 - 11 Washing

H₂O dist

4 x 400 ml

4 x 10 min

ambient

40 min

12 Silvering

0.25 % AgNO₃ /0.04% Formaldehyde (w/v)
(1000 mg AgNO₃/ 360 ul Formaldehyde (37%) in 400 ml)

400 ml

60 min

ambient

60 min

13-15 Washing

H₂O dist

3 x 400 ml

3 x 1 min

ambient

3 min

16 Developing

2.5 % Na₂CO₃ / 0.03% Formaldehyde / 0.00075% Sodium-Thiosulfate
(10 g NaCO₃/ 360 ul Formaldehyde / 150 ul Sodium-Thiosulfate (2%, fresh!) in 400 ml)

400 ml

5 - 7 min
visual control

ambient

6 min

17 Stopping

1.5% EDTA-Na
(6 g in 400 ml)

400 ml

40 min

ambient

40 min

18 Preserving/
Drying

wash 40 min with 10% Glycerol, then semi-dry 3 h on the support-film
(then roll on a polyester-sheet)

400 ml

3 h 40 min

ambient

3 h 40 min


	AgNO_{3, Na₂CO_{3 (water-free), Sodiumt}}hiosulfate are from Merck Ethanol, Acetic Acid, Formaldehyd (37%) and Glycerol are from Merck Abbr.: HAc = Acetic Acid, NaAc = Sodium Acetate

		EDC Electrophoresis Development & Consulting, Vor dem Kreuzberg 17, 72070 Tübingen (Germany)