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Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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	EDC’s SDS & 2D Silver Staining Protocol

		Ag-staining recipe for EDC’s ElphoGels SDS This special SDS staining recipe is necessary because EDC’s SDSGels use different buffer molecules to store and run them. For 2D-gels this staining should be mass-spectrometer compatible: No Glutardialdehyde in the Conditioner!

April 28, 2023

steps	reagent	volume	time (0.4-0.6 mm)
1 Fixing	40% (v/v) Ethanol / 10% (v/v) Acetic Acid, 1% Citric Acid (80 ml EtOH / 20 ml HAc + 2 g Citric Acid in 200 ml)	1 x 200 ml	60 min
2 Washing	H₂O dist	4 x 200 ml	4 x 10 min
3 Conditioning	30% (v/v) Ethanol; 0,325 % (w/v) Sodium Thiosulphate 165 mM Sodium Acetate; 2.5 % Glutardialdehyde (w/v), 0.07% (w/v) Benzenesulfonic acid (60 ml EtOH / 650 mg Thiosulfate / 4.5 g NaAc / 20 ml Benzenesulfonic stock sol., 20 ml Glutardialdehyde (25%) in 200ml)	200 ml	30 min
4 Washing	H₂O dist (place gel on the glas tray bottom surface side up)	4 x 200 ml	4 x 5 min
5 Silvering	0.2 % (w/v) AgNO₃ /0.04% (v/v) Formaldehyde (37%), 0.07% Benzenesulfonic Acid 8 ml AgNO3 - stock sol. / 20 ml Benzenesulfonic stock sol./ 260 µl Formaldehyde in 200 ml)	200 ml	45 min
6 Washing	H₂O dist	3 x 200 ml	3 x 1 min
7 Developing	2.5 % Na₂CO₃(w/v) 0.05% (w/v) Formaldehyde / 0.002% (w/v) Sodium-Thiosulfate (5 g NaCO₃ / 260 ul Formaldehyde (37%) / 200 µl Sodium-Thiosulfate (2%, fresh!) in 200 ml)	1 x 200 ml	2 - 4 min
8 Stopping/ Preserving	10% (v/v) HAc, 10% (v/v) Glycerol (20 ml HAc / 20 ml Glycerol in 200 ml)	200 ml	20 min
9 Drying	air dry , on the support-film then roll on a polyester-sheet	---	overnight


	Droplet-test: within 3 seconds

Stock Solutions
1. Benzenesulfonic (Merck 8.00468.0250), 0.7 % (10-fold solution) 1,4 g / 200 ml
2. AgNO₃: 5 % (20-fold solution), 5 g in 100 ml H₂O dist.
3. Na₂CO₃: 25% (10-fold) solution, 25 g (water-free!) in 100 ml H₂O dist.
4. Thiosulfate: 2%: 200 mg / 10 ml (prepare daily fresh!)
AgNO₃, _Na₂CO₃ (water-free), Sodiumthiosulfate are from Merck
Benzenesulfonic acid, Ethanol, Acetic Acid, Formaldehyd (37%), Glutardialdehyde, Citric Acid and Glycerol are from Merck Darmstadt (Germany)

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


	ElphoGel SDS 10% 25S stined with the recipe above


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