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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