Biology Reference
In-Depth Information
6. Electrode strips and sample application pieces (GE Healthcare).
7.
Filter paper (Whatman No. 1).
8.
Multiphor II (GE Healthcare).
9.
Refrigerated cooling bath.
10. Power supply for up to 2,000-3,000 V.
11. Transparent envelope.
12. Stapler.
2.4. Preparation
of SDS-PAGE Gels
1.
Separation gel buffer: 1.5 M Tris-HCl, pH 8.8 (see Note 7).
2.
Stacking gel buffer: 0.5 M Tris-HCl, pH 6.8, and 0.4% SDS
(w/v).
3.
T30C2.7 solution: 29.2% (w/v) acrylamide and 0.8% (w/v)
N
,
N
¢-methylenebisacrylamide, stored at 4-6°C.
4.
70% (v/v) glycerol.
5.
TEMED, stored at 4-6°C.
6.
10% (w/v) ammonium persulfate (not older than 1 week),
stored at 4-6°C.
7.
Glycerol-bromophenol blue solution: Mix 10 mL of water with
20 mL of glycerol and 25-50 mL of saturated bromophenol
blue solution. This solution is just for fi lling the void volumes
in the casting system and the bottom of the gel cassette; the
glycerol used here does not need to be of highest purity.
8.
Low-fl uorescence glass plates for the SE600 vertical electro-
phoresis chamber (18 × 16 cm; Hoefer Scientifi c Instruments).
9.
Spacers (1.5 mm thick).
10. 4-gel caster (including additional glass plates, fi ller sheets;
Hoefer Scientifi c Instruments).
11. Gradient maker (500 mL; Hoefer Scientifi c Instruments).
12. Magnetic stirrer.
13. Peristaltic pump.
14. Dual gel casting stand (Hoefer Scientifi c Instruments).
15. Preparative comb (for one strip and two single samples; Hoefer
Scientifi c Instruments).
2.5. Equilibration
1.
Equilibration solution (stock solution, for two strips): 2.5 mL
of stacking gel buffer (see Subheading
2.4
), 9 g urea, 10 mL of
70% (v/v) glycerol, 0.5 g SDS, 5 mL of water; each strip is fi rst
equilibrated in 5 mL of the stock solution supplemented with
100 mg DTT then incubated in 5 mL of the stock solution
with 125 mg iodoacetamide.
2.
Glass tubes with screw caps (20 mL; Schott, Jena, Germany).
3.
Laboratory shaker.
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