2

相关专题

 

his method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study.

Solutions (TIP: Use electrophoresis grade reagents to prepare the following solutions: )

A: 50 ml IEF Lysis Buffer

1. Add 21 g urea to 35 ml HPLC-grade H2O to a 50 ml Falcon tube (final concentration 7 M ).

2. Vortex vigorously for several minutes.

3. Add: 7.6 g Thiourea, 2 g Chaps, 0.5 g Mega 10, 0.5 g OBG, 250 µl Triton X-100, 0.25 g Tris, 0.4 g DTT, 500 µl Pharmalytes or IPG buffer pH 3-10, 500 µl -mercaptoethanol.

4. Add 10 µl tributylphosphine 2 mM , under the hood (2 mM final conc.)

5. Add Bromophenol Blue as indicator.

6. Check volume is 50 ml. Vortex until all is dissolved (or attach tube to a rotator).

7. Aliquot 1 ml into microfuge tubes. Store at -20°C .

B: 10X Electrophoresis Running Buffer (10 L ) 0.25 M Tris, 1.92 M glycine, 1M SDS

1. Add 300 g Tris-base, 1441 g glycine, and 10 g SDS to ~7 L HPLC-grade H2O.

2. Mix gently until dissolved. Bring volume to 10 liters.

C: 30% Acrylamide Stock (1 liter)

1. Add 292 g acrylamide and 8 g piperazine diacrylamide (PDA) to 700 ml HPLC-grade H2O, under the hood .

2. Stir to dissolve. Bring volume to 1 L .

3. Filter through 0.45 µm pore size filter. Store at 4°C in the dark.

D: Separating Acrylamide Gel

Below are the solution volumes required to prepare one 9-18% gradient gel. Prepare sufficient volume for the number of gels to be run.

Solution

Volume Units

9% gel

18% gel

1.5 M Tris-HCl, pH 8.8

ml

11.5

11.5

20% SDS

ml

0.23

0.23

30% Acrylamide

ml

14

28

TEMED

µl

11.7

11.7

10% APS

µl

117

117

HPLC-grade H2O

ml

20

6

Total

ml

45.8

45.8