Biology Reference
In-Depth Information
Table 2
Solutions for the preparation
of a native stacking PAGE gel
4% (mL)
Acrylamide (37.5/1)
1.33
6× Gel buffer
1.67
ddH 2 O
7.00
S
10.00
5. Add 10 mL of TEMED and 100 mL of the APS solution to the
solution and pipette the gel solution quickly between the glass
plates up to the top of the glass plate. The gel should polymerize
in about 15 min (see Note 11).
3.3. Native
Electrophoresis
(First Dimension)
1. After the stacking gel is polymerized, label the position of the
wells on the outer glass plate and remove the comb carefully
from the native gel. Assemble the electrophoresis apparatus.
2. Prepare the cathode running buffer by diluting 60 mL of the
5× cathode buffer with 240 mL of distilled water. Pour the 1×
cathode buffer into the upper chamber.
3. Rinse the wells with buffer using a 100-mL microsyringe to
remove the residues of acrylamide and entrapped air.
4. Underlay the samples into the wells using the microsyringe.
Rinse the microsyringe with buffer before applying a new sam-
ple (see Notes 12 and 13).
5. Add 3 mL of a 2% (w/v) bromphenol blue solution to an empty
well (see Note 14).
6. Dilute 100 mL of 10× anode buffer with 900 mL of distilled
water and pour the 1× anode buffer in the lower buffer
chamber.
7. Connect the tubes of the thermostatic circulator to the buffer
chamber. Start the thermostatic circulator. The electrophoretic
run is carried out at 4°C (see Note 15).
8. Assemble the electrophoresis unit completely and connect to a
power supply.
9. Set the power supply to 12 mA, 1,000 V and 24 W and start
the electrophoresis.
10. Stop electrophoresis when the bromphenol blue front has
reached the bottom of the separating gel (about 3.5 h).
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