Biology Reference
In-Depth Information
native IEF (Subheading 2.10 ) and fi rst dimension modSDS-
PAGE (Subheading 2.9 ).
2. Prepare the 4 % acrylamide stacking gel mixture
(Subheadings 2.9 and 2.10 )
3. Mount the gel cassette (1 mm spacer glass plate for the second
dimension or 0.75 mm for the fi rst dimension).
4. For the 1 mm gels add 2.5 mL (0.75 mm; 2 mL) solution I
and solution II to the two chambers of a gradient mixer with
the connecting channel closed ( see Note 11 ).
5. Add 13
μ
L 10 % APS (0.75 mm; 11
μ
L) and 1.3
μ
L TEMED
L) to each acrylamide mixture in the gradient
mixer and agitate using a magnetic stirrer.
6. Open the connecting channel of the gradient mixer and pump
the solution in the gel cassette until the mixer is empty (4 mL/
min).
7. Overlay the gel with 2-propanol and let the gel polymerize at
room temperature (~30 min).
8. Remove the 2-propanol from the top of the gel after polymer-
ization and wash it few times with water and remove the water.
9. Add 35
(0.75 mm; 1.1
μ
L TEMED to the 4 % acrylamide
stacking gel mixture. Pour the solution on top of the polymer-
ized resolving gel. For the second dimension fi ll gel solution
on the top of the resolving gel (~0.5 cm) and overlay with
2-propanol. For the fi rst dimension cast up to the top and
apply a comb. (polymerization ~ 15 min or over night at room
temperature).
10. Remove the 2-propanol, wash with water and wrap gel in water
soaked paper and foil. Store the gel at 4 °C until use.
μ
L 10 % APS and 5
μ
The native IEF-polyacrylamide gel should be prepared the day
before use. Do not keep it in storage.
3.6.2 Native IEF-Gel
1. Prepare the 7.5 % acrylamide gel mixture (7 mL): Mix 1.75 mL
AB-solution, 0.14 g CHAPS, 1.26 g urea, 350
L 40 % ampho-
lyte solution pH 3-10 and solve the components in a ultra-
sonic bath then fi ll up with water to 7 mL ( see Note 12 ).
2. Mount two gel cassettes (2× spacer glass plates 0.75 mm, 2×
front glass plates).
3. Add 10 % APS (70
μ
L), to the 7.5 % IEF
gel solution, mix it by pivoting and fi ll it into the casting cas-
sette until the liquid reaches the top and insert the comb.
4. Cover the gel with foil and let the gel polymerize over night
at RT.
μ
L) and TEMED (7
μ
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