Biology Reference
In-Depth Information
4. IPG sample buffer: Dissolve 100 mg dithiothreitol (DTT),
400 mg CHAPS, and 5.4 g urea in 6 mL of water and add
0.5 mL of alkaline carrier ampholytes (e.g., Pharmalyte pH
8-10.5, GE Healthcare; see Note 3). The buffer may be stored
in appropriate aliquots at −20°C.
2.2. IPG Preparation
1. Gels are cast on GEL-FIX for PAGE (260 × 125 mm; Serva,
Heidelberg, Germany) in a casting cassette (consisting of two
glass plates of 125 × 260 × 0.5 mm, to one of them a rubber
U-frame open at the longer side is attached; GE Healthcare).
The inner side of the U-frame glass plate is made hydrophobic
with Repel Silane (GE Healthcare); the procedure has to be
repeated at intervals to avoid any sticking of polyacrylamide.
2. pH gradients are established by Acrylamido buffer pKa 1
(Fluka, Buchs, Switzerland), Immobilines II pK 4.6, 6.2, 7.0,
8.5, 9.3 (GE Healthcare), 1 M acetic acid.
3. T30C4 solution: 28.8% (w/v) acrylamide and 1.2% (w/v) N ,
N ¢-methylenebisacrylamide (see Note 4). The T30C4 solution
is best stored in aliquots at −20°C.
4. 70% (v/v) glycerol.
5. TEMED.
6. 10% (w/v) ammonium persulfate, freshly made.
7. Gradient maker (15 or 30 mL; Hoefer Scientifi c Instruments,
San Francisco, CA, USA).
8. Magnetic stirrer.
9. Peristaltic pump.
10. Dryer/oven (for heating up to 50°C).
11. Glass tray to hold 1 L of solution.
12. Laboratory shaker.
13. Washing solution: 10 mL of glycerol in 1 L of water.
14. Ventilator or fan (cold air) to dry the gel.
15. Freezer (−20°C).
16. Plastic cover (transparent envelope) for storage.
2.3. First Dimension/
IEF
1. Re-swelling solution: 12 g urea, 0.5 g CHAPS, 37.5 mg DTT
dissolved in 16 mL of water, and 0.25 mL of ampholytes (see
Notes 5 and 6). The solution is stored in aliquots at −20°C.
2. DryStrip Aligner (GE Healthcare).
3. Glass plate.
4. Cover for the strips while swelling (glass tray/container) and a
quiet place for the strips to swell at room temperature.
5. n-Decan (Merck) as contact fl uid.
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