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 (cProtein = 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