Hoefer SE400 - Manual (Page 39)

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6. Resolving gel overlay
(0.375 M TrisCl, 0.1% SDS, pH 8.8, 100 ml)
1.5 M Tris-Cl, pH 8.8 (Solution #2) 0.375 M 25.0 ml
10% SDS (Solution #4)
3.5 mm 1.0 ml
Deionized H
2
O to 100.0 ml
Store up to 3 months at 4 °C in the dark.
—OR—
Water-saturated n-butanol
Shake n-butanol and deionized H
2
O in a separatory
funnel. Remove the aqueous (lower) phase. Repeat
this procedure several times. Use the upper phase.
—OR—
If an overlay interferes with the preferred protocol,
isolate the gel from atmospheric oxygen by placing a
preparative comb or resolving gel former on the gel.
7. 2X Sample treatment buffer
(0.125 M TrisCl, 4% SDS, 20% glycerol,
0.2 mM DTT*, pH 6.8, 10 ml)
0.5 M Tris Cl, pH 6.8 (Solution #3) 0.125 M 2.5 ml
10% SDS, 0.35 M (Solution #4)
0.14 M 4.0 ml
Glycerol (FW 92.09) 20% v/v 2.0 ml
Dithiothreitol (DTT) (FW 154.2) 0.2 mM 0.31 g
Bromphenol Blue (FW 691.9) 0.3 mM 2.0 mg
Deionized H
2
O to 10.0 ml
* or 2-mercaptoethanol* (FW 78.13) 2% v/v 0.2 ml
Divide into 1.0 ml aliquots and store at -40 °C to -80 °C for
up to 6 months.
—OR—
6X Sample treatment buffer
(0.35 M TrisCl, 10% SDS, 30% glycerol, 9.3% DTT,
pH 6.8, ~10 ml)
0.5 M TrisCl, pH 6.8 (Solution #3) 0.35 M 7.0 ml
SDS (FW 288.4) 0.35 M 1.0 g
Glycerol (FW 92.09) 30% v/v 3.0 ml
DTT (FW 154.2) 0.6 M 0.93 g
Bromphenol Blue (FW 691.9) 0.175 mm 1.2 mg
Divide into 1.0 ml aliquots and store at -70 °C.