Biology Reference
In-Depth Information
2.4. Isoelectric
Focusing (First
Dimension)
1. 2× Sample buffer (2× SB; 8 M urea, 4% CHAPS, 130 mM DTT,
2% (v/v) IPG buffer): Prepare fresh as required. Weigh 480 mg
of urea and 20 mg of DTT. Transfer to a graduated 1.5-mL
centrifuge tube. Add 200
μ
L of 20% CHAPS (see
Subheading
2.1
, item 2), 20
L of IPG buffer (pH 4-7 or 3-10)
(see Note 4), and make up to 1 mL with water (see Note 5).
μ
2.
Rehydration buffer (8 M urea, 4% CHAPS, 13 mM DTT, 1%
(v/v) IPG buffer): Prepare fresh as required. Weigh 480 mg of
urea and 2 mg of DTT. Transfer to a graduated 1.5-mL centri-
fuge tube. Add 200
L of IPG buffer
(pH 4-7 or 3-10) (see Note 4), and make up to 1 mL with
water (see Note 5).
μ
L of 20% CHAPS, 10
μ
3.
Cleaning solution for strip holder (see Note 6).
4.
Cover fl uid (see Note 7).
2.5. SDS-PAGE
(Second Dimension)
1.
10× Running buffer (250 mM Tris, 1.92 M glycine, 1% SDS):
Weigh 121.2 g of Tris base, 576.4 g of glycine, and 40 g of
SDS and transfer to a 5-L beaker. Make up to 4 L with water.
Do not adjust pH.
2.
Equilibration buffer (for one IPG strip) (EB; 6 M urea, 50 mM
Tris-HCl, pH 8.8, 30% glycerol, 2% SDS): Prepare fresh as
required and approximately 30 min before use. Weigh 18.2 g
of urea and 1 g of SDS. Transfer to a 100-mL beaker with a
magnetic stir bar. Add 1.68 mL of 1.5 M Tris-HCl, pH 8.8,
and 17.25 mL of 87% glycerol. Add water up to approximately
40 mL and mix to dissolve urea (see Note 8). Once dissolved,
make up to 50 mL with water.
3.
DTT-equilibration buffer (DTT-EB) for one IPG strip: Prepare
fresh as required. Weigh 0.1 g of DTT and transfer to a 50-mL
beaker. Add 20 mL of equilibration buffer prepared as described
above and mix until DTT is fully dissolved (see Note 9).
4.
Iodoacetamide-equilibration buffer (Iodo-EB) for one IPG
strip: Prepared fresh as required. Weigh 0.9 g of iodoacetamide
and transfer to a 50-mL beaker. Add 20 mL of equilibration
buffer prepared as described above and mix until iodoacet-
amide is fully dissolved (see Note 9).
5.
Saturated bromophenol blue solution: Add bromophenol blue
to a quarter full in a 1.5-mL tube. Add 1 mL of 30 mM Tris-
HCl, pH 8.5. Mix and let the undissolved bromophenol blue
settle. When in use, take an aliquot from the top without dis-
turbing the pellet.
6.
1% Agarose: Weigh 250 mg of low-melt agarose (Bio-Rad,
Hercules, CA, USA) and add 25 mL of water. Melt the agarose
by heating it on a low setting in a microwave. When fully dis-
solved, add 25
L of saturated bromophenol blue solution.
Aliquot into 1.5-mL tubes and store at room temperature.
μ
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