Hoefer SE400 Uživatelský manuál stáhnout pdf (Strana 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

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

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

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.

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Hoefer SE400 Uživatelský manuál Strana 39