Biology Reference
In-Depth Information
3.6.3
hrCNE Gradient Gel
Casting one 4-16 % hrCNE gradient gel (0.075 cm × 8 cm × 10 cm)
is exemplifi ed.
1. Prepare the 4 % acrylamide gel mixture (5 mL): Mix 0.625 mL
AB-solution, 1.67 mL hrCNE-GB (3×) (Subheading
2.12
,
item 1
) and 2.68 mL water.
2. Prepare the 16 % acrylamide gel mixture (5 mL): Mix 2.67 mL
AB-solution, 1.67 mL hrCNE-GB (3×), fi ll up to 5 mL with
glycerol.
3. Mount the gel cassette using two glass plates (1 mm × 0.75 mm
spacer glass plate, 1× front glass plate).
4. Add 1.75 mL 4 and 16 % acrylamide mixture to the two cham-
bers of a gradient mixer with the connecting channel closed
(
see
Note 11
).
5. Add 10 % APS (10
L), to each acryl-
amide mixture in the gradient mixer and agitate using a mag-
netic stirrer.
6. Open the connecting channel of the gradient mixer and pump
the solution in the gel cassette.
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 17.5
μ
L) and TEMED (1
μ
L TEMED to the rest of the
4 % acrylamide gel mixture (
see
step 1
), pour this solution on top
of the polymerized gradient resolving gel, and insert the comb.
μ
L 10 % APS and 1.75
μ
10. The sample gel polymerizes within 20 min.
11. Remove the comb and overlay with water and wrap it in water
soaked paper and foil. Store the gel at 4 °C until use.
3.7 Sample
Preparation for
Electrophoresis
1. Pellet membrane samples at 16,000 ×
g
for 90 min, remove
supernatant and resuspend the pellet in washing buffer. Shake
samples at 4 °C for 60 min.
2. Pellet washed membranes at 16,000 ×
g
for 90 min and remove
supernatant.
3.7.1 Membrane Washing
Before Solubilization
(Optional Step)
1. Prepare three aliquots of each samples (soluble fraction, micro-
somes, plasma membranes and tonoplast) in 1 mL tubes (
see
Note 8
): 20
3.7.2 Solubilization and
Preparation for
Electrophoresis
g protein for
membrane fractions for fi rst dimension PAGE; 50-75
μ
g for the soluble fraction and 50
μ
μ
g of
g protein for membrane fractions
for separation in 2D-PAGE.
2. Pellet membranes at 16,000 ×
g
for 90 min at 4 °C and remove
supernatant.
soluble proteins and 250
μ
