2D-Elektrophorese                           1.4.1998

IEF-Gel                                                                                 RT                                1 Tag

10 g     Harnstoff

6.5 ml   Aqua a.i.

3 ml     Rotiphorese Gel 30

1 ml     IGEPAL CA-630 (10% v/v in Aqua a.i.)

         oder Triton X 100 (10% v/v in Aqua a.i.)

entgasen (5 min)

20 µl    TEMED                            (Sigma T9281)

40 µl    APS (10% in Aqua a.i.)           (Sigma A3678)

15 mg    CHAPS                            (Sigma C3023)

960 µl   obige Gel-Lösung

28 µl    Ampholine pH 3.5 – 10            (Sigma A5174)

12 µl    Ampholine pH 5 – 7               (Sigma A

entgasen(1 min), Gele aufziehen

Probenpuffer                                                                     - 20°C                           o.A.

1.14 g   Harnstoff

40 µl    IGEPAL CA-630

30 µl    CHAPS                            (Sigma C 3023)

100 µ    Ampholyte (30% pH 5-7, 70% pH 3.5-10)

62 mg    DTE                              (Sigma D8255)

ad 2 ml mit Aqua a.i

Probenaufbereitung

1 Teil Probe (c_Protein = 1...2 mg/ml) +1 Teil Probenpuffer

Katholyt (oben)

343 ml   Aqua a.i.

7 ml     NaOH (1 mol / l )

entgasen(5 min)

 

Anolyt (unten)

4000 ml  Aqua dest.

2.77 ml  Phosphorsäure (85%)

 

Laufbedingungen

2 h      200 V 1 mA

ca. 16 h500 V 1 mA

3 h      900 V 1 mA

Transfer-Puffer                                                                  RT                                  o.A.

73 mg    Tris

10 mg    EDTA

3.75 ml  SDS 20%

1.5 ml   Glycerin

231 mg   DTE

0.75 ml  Bromphenolblau 0.2%

1.5 ml   Aqua a.i.

Stammlösung T (modifiziert) 1.5 M Tris-HCl, pH 9.18

76.96 g  Tris-Base

18.45 g Tris-HCl

ad 500 ml mit Aqua a.i.

SDS-Gel (kontinuierlich, für 4 Gele 200 x 200 x 1mm)         +4°C     10 Tage

86.5 ml  Rotiphorese Gel 30

53.2 ml Stammlösung T (Tris-HCl 1.5 mol/l, pH 8.8)

60 ml    Aqua a.i.

 

5 min entgasen

 

250 ml    TEMED                            Sigma T 9281

500 ml    APS (10% in Aqua a.i.)           Sigma A 3678

SDS-Gradientengel 7 …15% (für 4 Gele)   +4°C     10 Tage

 

5% T	7% T	15% T
13.3 ml 26.7ml 0.4ml 39.6ml	18.7ml 26.7ml 0.4ml 34.2ml	40.0ml 26.7ml 0.4ml 12.9ml	Rotiphorese Gel 30 Stammlösung T SDS 20% Aqua a.i.
entgasen 5 min
je 18.0ml je 50 ml je 12 ml			in den Gradientenformer APS 10% TEMED
Sammelgel (4% T)
2.0ml 1.9ml 75 ml 11.0ml			Rotiphorese Gel 30 0.5M Tris-HCl, pH 6.8 SDS 20% Aqua a.i.
entgasen 5 min
75 ml 15 ml			APS 10% TEMED

 

Laufpuffer    RT        Wochen

37.3 g   Glycin                           Sigma G 7403

9.1 g    Tris Base

30.0 g   SDS (= 150ml 20%ige Lösung)      Sigma L 4509

0.6 g    Natrium Acid (nur untere Pufferkammer)

auf 3000ml mit Aqua dest