Biology Reference
In-Depth Information
Table 1
Volumes for preparation of a native LDS-PAGE gel (Hoefer SE400)
“Light” solution,
3.5 % (v/v)
“Heavy” solution,
12 % (v/v)
Stacking gel,
3 % (v/v)
Solution
Acrylamide, 30 % (37.5:1)
1.95 mL
6.68 mL
0.5 mL
Gel buffer (6×)
2.784 mL
2.784 mL
0.833 mL
Glycerol, 50 %
1.67 mL
6.68 mL
0.5 mL
Water nanopure
10.296 mL
0.556 mL
3.167 mL
Total volume
16.70 mL
16.7 mL
5 mL
Degas 3 min
APS (10 %)
57.34
μ
L
47.8
μ
L
25
μ
L
TEMED
11.47
μ
L
4.78
μ
L
5
μ
L
chamber 1 (chamber 2). Ensure that the “heavy” solution in
chamber 1 is placed at the center of the magnetic stirrer and
stirring bar is free to turn (
see
Note 9
and Table
1
).
8. Set the tube-pump speed to 11.5 mL/min (
see
Note 10
).
9. Add catalysts APS and TEMED for initiation of the acrylamide
polymerization reaction (
see
Table
1
).
10. Start pump.
11. Open connection between chamber 1 and 2.
12. Let the solutions run between the glass plates.
13. Overlay the cast gel with water-saturated isobutanol. Allow gel
to polymerize for at least 90 min.
14. Rinse the gradient mixer immediately with distilled water to
prevent the polymerization of residual gel solution in the tubes
and the gradient mixer tubing.
1. After polymerization of the separating gel, remove isobutanol
by washing with distilled water.
2. Dry area above separating gel with Whatman paper. Do not
touch separation gel surface.
3. Clean comb with denatured ethanol (100 %).
4. Add APS and TEMED to stacking solution and pipette onto
separation gel. Fill sandwich to the top of the glass plate.
5. Insert dried comb in stacking solution. Make sure no bubbles
are trapped at front of comb or between teeth and align comb
teeth ends parallel to separation gel (
see
Note 11
).
6. Let gel polymerize for at least 30 min (
see
Note 12
).
3.5 Casting
of Stacking Gel
