Laboratorio Bellinzona, Neurologie Tübingen, Labor Limbach Heidelberg

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1 Report and Summary from Dr. HP Schickle Tübingen, Laboratorio Bellinzona, Neurologie Tübingen, Labor Limbach Heidelberg Protocol of the experiments in EDC s lab to solve the problem with the Burning Cathode and the Staining Gap. Burning Cathode final voltage too high for salty samples: Cathode is Burning LOT #093829G cathode is not anymore burning by reducing the final voltage from 1900 V to 1500 V. strong Staining Gap LOT #093829G Step Volt ma W time new running conditions EDC Electrophoresis Development & Consulting, manuals: -> downloads 1

2 Staining Gap strong Staining Gap LOT #093829G Staining Gap does also appear (a little bit) in the Neurologie Tübingen LOT #093829G Staining Gap does not appear everywhere (example from Hannover). EDC Electrophoresis Development & Consulting, manuals: -> downloads 2

3 New Mixes to be tested to avoid the staining gap Mix #6. Mix #7. EDC Electrophoresis Development & Consulting, manuals: -> downloads 3

4 Different silver-staining recipes There are different recipes in different laboratories. EDC will test them one after the other. The most important three solution are: Fix #1, Incubation, Silvering and the Developer. Fix #1 Solution EDC Tübingen Heidelberg Bellinzona* TCA 20% 11.5% 20% 20% Additives 30% MeOH 3.5% Sulphosalicylic Acid Incubation Solution EDC Tübingen Heidelberg Bellinzona* Glutardialdehyde 2.5% 6% 2.5% 2.5% Additives 1.75% NaAc Silvering Solution EDC Tübingen Heidelberg Bellinzona* Ag 0.125% 0.4% 0.125% NH % 0.5% 0.25% 0.19 NaOH 0.02M 0.01M 0.02M 0.02M Developer Solution EDC Tübingen Heidelberg Bellinzona* Formaldehyde 100µl/200ml 120µl/200ml 100µl/200ml 100µl/200ml Citric Acid % 0.005% 0.005% % *Staining in Hoefer s autostainer EDC Electrophoresis Development & Consulting, manuals: -> downloads 4

5 EDC-Manual, LOT #093829G, left: M eoh, right: Rotisol (see page 10), background, slight staining gap. Time: ~4.5 h (with overnight step) Tübingen, LOT #093829G: Clear background but a staining gap appears. Time: 4 h (with overnight step) Heidelberg, LOT #093829G: N o staining gap, background. Time: ~3.5 h (without overnight step) EDC Electrophoresis Development & Consulting, manuals: -> downloads 5

6 Ammoniacal Silver-Staining Recipe EDC-manual This is the normal, proposed (Dr. U.Wurster, Hannover) staining procedure described in the manual IEFGel Also on this page of EDC s website: It did function with both: MeOH and Rotisol*, see pictures below. * Rotisol: Roth (Karlsruhe, Germany #7917.1): 95% EtOH, 5% Aceton (low price product) LOT #093829G ammonical silver using MeOH EDC Electrophoresis Development & Consulting, manuals: ammonical silver using Rotisol* (EtOH) -> downloads 6

7 Alternative Ammoniacal Silver-Staining Recipe from Heidelberg. In this laboratory they never had a Staining Gap Solutions Fix I: 20% TCA Fix II: 50% EtOH, 10% Essigsäure Fix III: 2,5% Glutardialdehyd Waschlösung: 5% Rotisol (= 95% Ethanol + 5% Aceton) Entwickler: 100 µl 10% Zitronensäure µl 37% Formaldehyd auf 200 ml Aqua dest. Bei der Entwicklerlösung steht bei unserer Vorschrift das in Klammern, in der Routine gelten aber viel eher die Zeiten die nicht in Klammern stehen, drei Minuten wären schon zu viel. Das Protokoll gilt für 40s und 80s Gele. Verdünnt werden die Seren für das 40s Gel 1:201 und für das 80s Gel 1:401 und aufgegeben wird in beiden Fällen 10µl. Solution [min] Fix I Fix II Waschlösung Fix III Waschlösung Aqua dest. Silberfärbung Entwickler Stopp x ,5 2 (3 10) Overnight IEFGel S Heidelberg: no staining gap IEFGel S Heidelberg: no staining gap. EDC Electrophoresis Development & Consulting, manuals: -> downloads 7

8 Alternative Ammoniacal Silver-Staining Recipe from Tübingen LÖSUNGEN FIXIERLÖSUNG g Trichloressigsäure 35,5 g Sulfosalicylsäure-Dihydrat 300 ml Methanol 700 ml Aqua dest. FIXIERLÖSUNG ml Methanol 100 ml Eisessig 400 ml Aqua dest. FIXIERLÖSUNG ml Glutardialdehyd 600 ml Aqua dest. WASCHLÖSUNG 200 ml Methanol 1800 ml Aqua dest. SILBERFÄRBUNG (frisch ansetzen) 31,5 ml NaOH 0,1 m 4,5 ml Ammoniak 6 ml Silbernitratlösung auffüllen mit 300 ml Aqua dest. Silbernitratstammlösung: (im Kühlschrank lagern) 10 g Silbernitrat + 50 ml Aqua dest. ZITRONENSÄURELÖSUNG (frisch ansetzen) 30 ml Zitronensäurelösung auffüllen auf 300 ml Aqua dest. Zitronensäurestammlösung: (im Kühlschrank lagern) 0,5 g Citronensäure-1-Hydrat 1000 ml Aqua dest. STOPPlÖSUNG (frisch ansetzen) 30 ml Essigsäure auffüllen auf 300 ml Aqua dest. KONSERVIERUNGSLÖSUNG 500 ml Methanol 160 ml Eisessig 200 ml Glycerin auffüllen auf 2 l Aqua dest. FIXIERLÖSUNGEN 1, 2, 3 UND KONSERVIERUNGSLÖSUNG ZURÜCKSCHÜTTEN UND MAX. 3 MONATE VERWENDEN EDC Electrophoresis Development & Consulting, manuals: -> downloads 8

9 PROTOKOLL FIXIERUNG UND FÄRBUNG DER IEF FIXIERLÖSUNG 1 15 Minuten FIXIERLÖSUNG 2 30 Minuten WASCHLÖSUNG 30 Minuten FIXIERLÖSUNG 3 30 Minuten WASCHLÖSUNG 30 Minuten alles auf dem Schüttler! ÜBER NACHT IN AQUA DEST. LEGEN, OHNE ZU SCHÜTTELN! SILBER LÖSUNG 30 Minuten AQUA DEST. 15 Minuten ENTWICKLERLÖSUNG 3-5 Minuten STOPPLÖSUNG 5 Minuten KONSERVIERUNGSLÖSUNG 1 Stunde GEL AUF EINER FLACHEN UNTERLAGE MIT DER GELSEITE NACH OBEN EINEN TAG TROCKNEN, DANACH FOLIE AUFROLLEN UND BESCHRIFTEN. Staining recipe from Tübingen EDC Electrophoresis Development & Consulting, manuals: -> downloads 9

10 Automated ammoniacal silver-staining from Bellinzona Recipe is from the EDC manual. The staining itself is done with Hoefer s Autostainer. staining in Bellinzona: strong staining gap, time in Autostainer : 8h 45 min EDC recommends to do one manual staining to control the functionality of the apparatus: The tubings and the switching-valve are consumables (!) and have to be renewed from time to time: nomally once per year! EDC Electrophoresis Development & Consulting, manuals: -> downloads 10

11 Optimization on EDC s manual silver staining The two targets are: 1. Reduced background as in the staining of Tübingen. Fix 1: Reduction of TCA from 20% to 15% and addition of 30% MeOH. 2. Avoiding the staining gap by reducing the time of the washing steps as in Heidelberg. 3. Silvering and Developer as in EDC s manual. The Silvering step is shorter (Tübingen, Heidelberg). Newly introduced: in red colour Step Solution Time Recipe from Fix 1 15% TCA 30% MeOH 30 min Tübingen, EDC Rinse 1 50% MeOH 10% HAc 30 min EDC Rinse 2 5% MeOH 7% HAc 20 min EDC Incubation 2.5% Glutardialdehyde 20 min EDC Washing H 2 O dist 3 x 15 min* EDC, Heidelberg Silvering 0.125% AgNO % NH M NaOH 30 min EDC, Tübingen, Heidelberg Developer Stop 100µl/200ml Formaldehyde % Citric Acid 1% HAc 10% Rotisol* 3-5 min EDC 20 min Heidelberg, EDC Preserving 5% Glycerol 20 min EDC Total Time: 3 h 40 min, with no overnight step. Air-dry 24 h. * Overnight step in H 2 O * Rotisol: Roth (Karlsruhe, Germany #7917.1): 95% EtOH, 5% Aceton (low price product) EDC Electrophoresis Development & Consulting, manuals: -> downloads 11

12 Optimized Silver Staining Recipe with overnight step EDC Electrophoresis Development & Consulting, manuals: -> downloads 12

13 without overnight step EDC Electrophoresis Development & Consulting, manuals: -> downloads 13